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AGRfC. 
LIBRARY 


FACTORS    INFLUENCING   THE    PATHOGENICITY 
OF  HELMINTHOSPORIUM  SATIVUM 


A  THESIS 

SUBMITTED  TO  THE  FACULTY  OF  THE  GRADUATE 
SCHOOL  OF  THE  UNIVERSITY  OF  MINNESOTA 


BY 


LOUISE  DOSDALL,  M.A. 


IN  PARTIAL  FULFILMENT  OF  THE  REQUIREMENTS  FOR  THE 
DEGREE  OF  DOCTOR  OF  PHILOSOPHY 


JUNE,  1922 


FACTORS    INFLUENCING    THE    PATHOGENICITY 
OF  HELMINTHOSPORIUM  SATIVUM 


A  THESIS 

SUBMITTED  TO  THE  FACULTY  OF  THE  GRADUATE 
SCHOOL  OF  THE  UNIVERSITY  OF  MINNESOTA 


BY 

LOUISE  DOSDALL,  M.A. 
11 


IN  PARTIAL  FULFILMENT  OF  THE  REQUIREMENTS  FOR  THE 
DEGREE  OF  DOCTOR  OF  PHILOSOPHY 


JUNE,  1922 


Sis  74  / 

CONTENTS 


AGRIC.  Pa_ 

UBRARY 

Introduction   and   historical    review  ........................................  3 

Problem    ...............................................................  •  4 

Methods    ...............................    ................................  5 

Source  of  pathogene  ..................................................  5 

Selection  of  host  varieties  .............................................  6 

Check  plants    ...............................  .........................  6 

Specific  identity  of  the  pathogene  .....................................  7 

Temperature  relations  ....................................................  J6 

Growth  of  fungus  on  potato  dextrose  agar  ............................  16 

Spore    germination    ..................................................  18 

Effect  of  hydrogen-ion  concentration  and  temperature  on  spore  germination  21 

Infection     ...........................................................  25 

Influence  of  type  of  soil  ..................................................  27 

Influence  of   soil   moisture  .........  .......................................  31 

Influence  of   soil    fertility  ................................  .  ................  39 

Comparison  of  several  root-rot  causing  organisms  .........................  4° 

Summery    and    conclusions  ................................................  44 

Literature   cited    .........................................................  47 

ILLUSTRATIONS 

Fig.  i.     Types    of    curves    obtained    from    measuring    length    of    snores    of 

Helminthosporium  sativum  produced  on  potato  dextrose  ...........  n 

Fig.  2.  Length  of  spores  produced  on  potato  dextrose  agar  at  various 

temperatures     ..................................................  15 

Fig.  3.     Length  of  spores  produced  on  different  substrata  at  24°  C  ............  16 

Fig.  4.     Growth  of  H.  sativum  on  potato  dextrose  agar  in  Petri  dishes  .......  17 

Fig.  5.  Daily  rate  of  growth  of  H.  sativum  on  potato  dextrose  agar  in  Petri 

dishes     ........................................................  18 

Fig.  6.  Percentage  germination  of  spores  in  phosphoric  acid-potassium 

hydroxide  solutions  of  various  hydrogen-ion  concentrations  .......  23 

Fig.  7.  Percentage  germination  of  spores  in  Czapek's  solution  minus  the 

sugar  at  various  hydrogen-ion  concentrations  .....................  24 

Plate  I.  Helminthosporium  sativum  P.K.B.  grown  on  potato  dextrose  agar 

at    different    temperatures  ........  '  ...............................  49 

Plate  II.  Helminthosporium  sativum  P.K.B.  grown  on  potato  dextrose  agar 

at    different    temperatures  .......................................  50 

Plate  III.  Marquis  wheat  showing  effect  of  Helminthosporium  root-rot  in 

different  soils    .................................................  51 

Plate  IV.  Lion  barley  plants,  3  weeks  old,  growing  on  soils  inoculated  with 

various  organisms    .............................................  52 

Plate  V.  Lion  barley  plants  3  weeks  old  showing  effect  of  soil  organisms 

on  development  of  root  systems  .................................  53 

Plate  VI.  Lion  barley  pl?nts,  3^  weeks  old,  showing  effect  of  root  infection 

by  H.  sativum  P.K.B  ....................................  ........  54 


FACTORS    INFLUENCING    THE    PATHOGENICITY    OF 
HELMINTHOSPORIUM    SATIVUM 

By  LOUISE  DOSDALI^ 

INTRODUCTION  AND  HISTORICAL  REVIEW 

In  1910  Pammel,  King  and  Bakke  (9)  described  a  new  Helmin- 
thosporium disease  of  barley  which  they  called  "late  blight."  The 
causal  organism  was  named  Helminthosporium  sativum  n.  sp.  Pammel, 
et  al.,  had  observed  the  disease  in  Iowa  in  1907  and  1908.  In.  1909  it 
was  very  serious.  In  the  same  year,  they  report  that  it  was  also  found 
in  South  Dakota,  Minnesota,  and  Saskatchewan.  These  authors  de- 
scribe the  disease  as  follows :  "Brown  spots  of  irregular  outline  occur 
upon  the  leaves  causing  them  to  turn  brown.  The  leaves  are  easily 
broken  up,  and  in  some  cases  completely  destroyed.  The  disease  also 
occurs  upon  the  glumes,  spikelets  and  seed.  The  straw  at  harvest 
is  dull  brown,  and  instead  of  standing  erect  becomes  a  tangled  mass. 
The  date  of  ripening  of  the  grain  corresponds  with  the  time  of  full 
development  of  the  late  blight."  They  observed  that  there  was  con- 
siderable difference  in  varietal  susceptibility,  the  degree  of  .infection 
ranging  from  o  to  100  per  cent.  Late  blight  was  considered  the  most 
serious  disease  of  barley  in  Iowa. 

In  1913  A.  G.  Johnson  (7)  differentiated  clearly  the  three  Helmin- 
thosporium diseases  of  barley  in  Wisconsin,  and  he  designated  the  one 
caused  by  H.  sativum  P.K.B.,  the  "American  blotch  disease." 

Louise  Stakman  (n),  in  1920,  showed  that  a  Helminthosporium 
similar  to  the  organism  described  as  H.  sativum  by  Pammel,  King 
and  Bakke,  but  isolated  from  various  parts  of  diseased  wheat  and  rye 
plants,  was  capable  of  causing  a  serious  seedling  blight  of  these  hosts, 
and  could  also  attack  the  older  parts  of  the  plants,  namely,  the  leaves, 
nodes,  culms,  roots,  glumes,  and  grains.  In  addition  to  wheat  and 
rye,  successful  infections  were  obtained  on  barley  and  a  number  of 
grasses.  In  the  spring  and  early  summer  of  1919,  serious  attacks  of 
seedling  blight  caused  by  Helminthosporium  occurred  in  practically  all 
the  wheat-growing  regions  of  Minnesota. 

F.  L.  Stevens  (12),  also  in  1920,  reported  that  a  species  of 
Helminthosporium  was  constantly  associated  with  foot  rot  disease  of 
wheat  in  Madison  County,  111.  Inoculations  with  the  organism  gave 
positive  results.  He  concluded  that  Helminthosporium  was  the  cause 
of  the  disease. 

1  The  writer  wishes  to  express  her  appreciation  to  Dr.  E.  C.  Stakman,  under  whom  the 
work  was  done,  for  advice  and  criticism,  and  to  Mr.  M.  N.  Levine  for  his  helpful  criticism  in 
the  presentation  of  the  biometrical  studies. 


£43978 


'*  4  TECHNICAL  BULLETIN  17 


'''•In  Jaw4*£:  1922,  Hamblin  (5)  reported  a  Helminthosporium  foot- 
rot  disease  of  wheat  in  New  South  Wales,  Australia.  The  disease 
symptoms  are  very  similar  to  those  of  the  true  take-all  caused  by 
Ophiobolus  graminis  Saccardo,  but  there  are  distinguishing  characters. 
Hamblin's  description  of  the  foot-rot  in  Australia  corresponds  very 
closely  with  that  of  Mrs.  Stakman  and  of  F.  L.  Stevens.  His  descrip- 
tion of  the  poorly  developed  root  system  with  an  abnormal  develop- 
ment of  root  hairs  close  to  the  culm,  giving  the  dead  or  dying  root  a 
"fuzzy"  appearance,  and  the  frequent  growth  of  secondary  roots  above 
the  first  node  of  the  affected  straws,  applies  equally  well  to  conditions 
observed  in  Minnesota  during  1921.  In  Hamblin's  opinion,  the  Hel- 
minthosporium  disease  was  responsible  for  far  more  damage  in  1921  in 
Australia  than  was  the  better  known  take-all. 

In  recent  years,  a  foot-rot  disease  of  cereals,  particularly  wheat, 
rye,  and  barley,  has  been  destructive  in  certain  localities  in  Minnesota. 
This  was  especially  true  on  certain  peat  lands  in  Anoka  and  St.  Louis 
counties  and  on  some  of  the  sandy  soils  in  Anoka,  Nicollet,  and 
Mahnomen  counties.  A  Helminthosporium  of  the  sativum  type  has 
been  consistently  isolated  from  the  diseased  plants.  This  organism 
is  very  widely  distributed  throughout  the  cereal  growing  region.  The 
severity  of  its  attack  apparently  must  be  greatly  influenced  by  eco- 
logical conditions.  In  order  to  obtain  more  detailed  and  accurate  in- 
formation concerning  these  conditions,  a  study  of  the  physiology  of 
the  fungus,  to  the  extent  of  its  possible  correlation  with  the  pathogenicity 
under  given  conditions,  was  undertaken. 

PROBLEM 

In  this  study  attention  was  directed  primarily  to  the  root-  and  foot- 
rots  caused  by  H  sativum.  Little  attention  was  given  to  secondary  in- 
fections on  leaves  and  heads.  The  soil  environment  was,  therefore, 
of  chief  concern.  In  analyzing  the  factors  which  might  influence  the 
development  of  a  disease  of  this  type,  temperature,  moisture,  and 
acidity  would  affect  the  growth  of  both  the  pathogene  and  the  host, 
and  possibly  also  the  reaction  between  the  two.  The'  vigor  of  the  host 
conceivably  might  greatly  influence  the  development  of  a  disease  caused 
by  a  facultative  parasite,  such  as  H.  sativum.  The  type  of  soil  in 
which  they  grew  and  the  available  nutriment  might,  therefore,  change 
the  balance  between  host  and  pathogene.  It  is  difficult  to  separate 
and  analyze  the  individual  factors,  because  certain  combinations  intro- 
duce various  complexities  which  are  difficult  to  interpret. 

The  following  phases  of  the  problem  were  investigated  especially: 

i.  Relation  of  temperature  to  the  growth  of  the  fungus,  to  spore 

germination,  to  infection,  and  to  the  development  of  the  disease. 


PATHOGENICITY  OF  H.  SATIVUM  5 

2.  Relation  of  hydrogen-ion  concentration  and  temperature  to  Fpore 
germination. 

3.  Development  of  the  disease  in  various  types  of  soil. 

4.  Influence  of  soil  moisture  on  the  development  of  the  disease. 

5.  Influence  of  soil  fertilization  on  the  development  of  the  disease. 

6.  Comparison  of  several  root-rotting  organisms. 

7.  Morphological  variation  in  the  fungus  with  regard  to  its  specific 
identity. 

METHODS 
SOURCE  OF  PATHOGENE 

During  the  spring  and  summer  of  1920,  tissue  cultures  were  made 
from  lesions  caused  by  Helminthosporium  on  cereals  and  grasses. 
Twenty-two  strains  (isolations  from  various  parts  of  different  hosts 
or  from  different  localities)  of  the  sativum  type  were  obtained  from 
the  roots,  stems,  nodes,  leaves,  and  kernels  of  barley;  from  the  roots, 
stems,  leaves,  and  kernels  of  wheat ;  and  from  leaf  spots  of  various 
grasses.  Material  was  obtained  from  Anoka,  Clay,  Mahnomen,  Nicol- 
let,  Ramsey,  and  St.  Louis  counties  in  Minnesota;  from  Tennessee, 
and  from  Spruce  Grove  and  Edmonton,  Alberta. 

Seven  of  these  strains  were  selected  for  preliminary  inoculation 
experiments.  As  a  virulent  root-rotting  organism  was  desired,  only 
soil  inoculations  were  made.  Four-inch  pots  filled  with  soil  were  treated 
with  live  steam  for  two  hours  on  each  of  three  successive  days.  Six 
pots  of  such  soil  were  inoculated  with  each  of  the  various  strains  of 
Helminthosporum.  For  this  purpose,  spores  were  scraped  from  the 
surface  of  potato  dextrose  agar  cultures  and  mixed  with  water.  The 
suspension  of  spores  was  poured  over  the  soil,  and  the  pots  were  in- 
cubated for  several  days.  Three  pots  which  had  been  inoculated  with 
each  strain  were  then  sowed  with  Marquis  wheat  and  three  with 
Manchuria  barley  (Minn.  105).  Some  infection  was  obtained  in  each 
case,  on  both  the  leaves  and  the  roots.  (The  check  plants  were  slightly 
infected,  as  the  seed  had  not  been  treated.)  The  plants  inoculated 
with  strain  82a,  however,  were  decidedly  more  heavily  attacked  than 
the  others.  This  was  especially  true  of  the  barley  plants.  A  Helmin- 
thosporium of  the  sativum  type  was  re-isolated  from  lesions  on  both 
the  barley  and  the  wheat.  A  single  spore  culture  was  then  made  from 
the  original  82a  culture,  and  all  subsequent  work  was  done  with  this 
single  spore  strain. 

Culture  82a  was  originally  isolated  from  the  darkened  base  of 
badly  stunted  barley  plants  sent  to  the  laboratory  from  the  peat  plots 
on  the  Fens  experimental  field,  St.  Louis  County,  Minn.,  in  the  summer 


6  TECHNICAL  BULLETIN  17 

of   1920.     A  similar  Helminthosporium  was  isolated  from  the  nodes, 
sheaths,  and  blades  of  the  same  plants. 

In  addition  to  H.  sativum,  Alternaria  was  frequently  obtained  from 
blackened  kernels  and  nodes;  a  pink  Fusarium  was  sometimes  found 
on  the  base  of  the  stem  and  roots ;  and  Helminthosporium  teres  Sacc. 
was  occasionally  isolated  from  the  leaves  and  stems. 

SELECTION  OF  HOST  VARIETIES 

In  all  experiments,  the  effect  of  the  fungus  on  wheat  and  barley 
only  was  studied.  In  most  cases  where  barley  was  tested,  both  Man- 
churia (Minn.  105)  and  Lion  (Selection)  were  used.  Manchuria  is 
the  barley  most  commonly  grown  in  Minnesota.  It  is  somewhat  re- 
sistant to  H.  sativum,  as  shown  by  the  work  of  Pammel,  King  and 
Bakke  (9),  of  Hayes  and  Stakman  (6),  and  of  Christensen  (3).  For 
this  reason  it  was  used  in  the  breeding  work  of  Hayes  and  Stakman. 
It  was  crossed  with  the  smooth-awned  Lion,  which  is  very  susceptible 
to  Helminthosporium,  in  an  attempt  to  obtain  a  high  yielding,  smooth- 
awned,  resistant  variety. 

Marquis  (Minn.  1239)  was  used  in  most  of  the  experiments  with 
wheat.  In  some  cases  Haynes  Bluestem  (Minn.  169)  also  was  used. 

CHECK  PLANTS 

Since  it  is  difficult  to  obtain  seed  entirely  free  from  Helmintho- 
sporium, especially  in  susceptible  varieties,  it  was  necessary  to  treat 
the  seed  in  order  to  reduce  infection  in  the  check  plants  to  a  minimum. 
Silver  nitrate  was  found  to  be  the  most  useful  disinfectant  because 
the  seed  coats  of  both  barley  and  wheat  are  impermeable  to  it  (10), 
and  the  seed  can  be  soaked  for!  a  long  time  in  the  solution  without 
being  injured.  It  also  is  more  effective,  especially  against  Helmintho- 
sporium, than  mercuric  bichloride.  For  experimental  purposes,  the 
method  of  seed  treatment  followed  was  essentially  that  recommended 
by  Schroeder  (10).  The  seed  was  dipped  in  50  per  cent  alcohol  to 
remove  the  air  from  the  surface,  soaked  over  night  in  N/ioo  silver 
nitrate  solution,  dipped  in  a  dilute  sodium  chloride  solution  to  precipi- 
tate as  insoluble  silver  chloride  the  silver  nitrate  remaining  on  the 
surface  of  the  seed,  washed  thoroly  in  running  tap  water,  and  dried. 
Such  treatment  reduced  the  germination  of  Lion  barley  from  90  per 
cent  to  78  per  cent,  and  of  Marquis  wheat  from  99  per  cent  to  97 
per  cent. 


PATHOGENICITY  OF  H.  SATIVUM  7 

SPECIFIC  IDENTITY  OF  THE  PATHOGENE 

Three  species  of  Helminthosporium  are  known  to  occur  on  barley 
in  the  United  States.  These  are  readily  distinguished  on  the  host  by 
the  symptoms.  H.  gramineum  Rabh.  causes  the  systemic  stripe  disease 
characterized  by  long,  narrow,  yellowish  to  brownish  spots  on  the 
leaves  and  sheaths.  Many  spots  often  coalesce  to  form  parallel  stria- 
tions  which  run  more  or  less  the  entire  length  of  the  blade  and  often 
down  the  sheath.  Eventually  the  leaves  may  be  reduced  to  shreds. 
H.  teres.  Sacc.  and  H.  sativum  P'.K.B.  both  cause  local  lesions  which 
are  characterized  by  peculiar  blotches  on  the  leaves.  H.  teres  causes 
the  European  blotch  or  net  blotch  disease.  The  spots  are  yellowish 
brown  in  color,  irregular  in  shape,  and  are  scattered  on  the  leaves. 
When  held  to  the  light,  a  characteristic  net  work  is  apparent.  H. 
sativum  causes  the  spot  blotch  disease  characterized  by  irregular  red- 
dish brown  spots  on  the  leaves.  The  spots  are  usually  longer  than 
they  are  broad,  and,  when  abundant,  may  tend  to  form  stripes. 

These  three  species  also  may  be  distinguished  readily  by  their 
growth  on  potato  dextrose  agar.  H.  gramineum  grows  slowly,  forms 
a  fluffy,  aerial  mycelium  which  does  not  sporulate  (at  least  not  readily), 
and  usually  gives  the  medium  a  reddish  or  purplish  tinge.  H.  teres 
also  grows  rather  slowly.  The  mycelium  grows  very  close  to  the 
surface  of  the  agar.  The  color  of  the  reverse  side  of  the  colony  is 
greenish  black.  Grayish  white  tufts  of  mycelium  are  formed  irregu- 
larly on  the  surface  of  the  colony.  Cylindrical,  thin-walled  spores  are 
formed,  but  usually  they  are  not  abundant.  In.  contrast  to  both  these 
species,  H.  sativum  grows  very  readily  and  sporulates  abundantly, 
forming  a  flat,  black  or  greenish  black  colony  on  agar.  The  abundance 
of  conidia  gives  the  surface  a  powdery  appearance.  Organisms  similar 
to  the  one  isolated  from  typical  barley  spot  blotch  have  been  isolated 
hundreds  of  times  by  workers  in  this  laboratory  from  various  parts 
of  barley,  wheat,  and  rye  plants,  and  from  numerous  grasses. 

Pammel,  King  and  Bakke  (9)  described  the  spores  as  cylindric 
in  shape,  straight  or  curved,  slender,  widest  at  the  middle,  from  105 
to  130  microns  in  length  by  15  to  20  microns  in  width,  pale  greenish 
gray  to  dark  brown  in  color,  with  7  to  14  cells.  Later  workers  have 
found  much  shorter  spores,  altho  observations  on  shape  agree  fairly 
well.  Johnson  (7)  states  that  the  spores  are  narrowly  spindle-shaped, 
usually  more  or  less  curved.  Mrs.  Stakman  ( 1 1 )  describes  the  spores 
of  the  organism  with  which  she  worked  as  either  straight  or  curved, 
dark  blue-green  to  brown  in  color,  averaging  41  by  20  microns  in/  size, 
and  containing  from  3  to  8  septa.  Two  types  were  isolated  from  dis- 
eased wheat :  one  a  fuscous  type  measuring  35  by  22  microns  and 
containing  from  3  to  4  septa;  the  other  straw-colored  to  fuscous, 


8  TECHNICAL  BULLETIN  17 

measuring  60  by  20  microns,  and  containing  from  4  to  7  septa.  Both 
of  the  latter  are  described  as  elliptical  in  shape. 

Stevens  (12)  makes  the  following  statement  regarding  the  form 
causing  the  foot-rot  of  wheat:  "The  spores,  observed  as  grown  on 
autoclaved  wheat  leaves  or  stems  in  humid  air,  are  from  24  to  122 
microns  long,  the  majority  of  them  falling  within  the  limits  80  to  90 
microns,  with  septa  or  pseudo-septa  varying  from  o  to  13,  usually 
5  to  10.  The  spores  are  usually  typically  thickest  in  the  region  about 
midway  between  the  base  and  the  middle  point  of  the  spore,  approach- 
ing a  narrow  or  broadly  elliptical  shape,  tapering  somewhat  toward 
each  end.  They  possess  an  outer  dark  wall  that  is  thin  and  extremely 
fragile  and  an  inner,  colorless,  thick  wall  that  is  frequently  soft  and 
gelatinous  .  .  .  The  spores  usually,  perhaps  always,  germinate 
either  from  one  or  both  ends,  not  laterally,  and  are  functionally  only 
one-celled." 

After  making  a  large  number  of  isolations  from  Helminthosporium 
lesions  on  barley,  wheat,  and  rye,  great  variations  were  found  in  the 
size  of  the  spores  of  the  various  cultures,  altho  they  resembled  each 
other  more  or  less  in  shape  and  color.  In  order  to  find  out  just  what 
variations  might  be  expected  in  one  strain,  as  a  guide  to  the  interpreta- 
tion of  the  species,  a  single  spore  was  again  isolated  from  culture  82a 
and  a  biometric  study  was  made  of  the  spores  produced  under  various 
conditions. 

The  single  spore  was  planted  on  a  potato  dextrose  agar  slant  and 
incubated  at  24°  C.  for  ten  days.  Transfers  were  then  made  to  potato 
dextrose  agar  and  to  ripe  autoclaved  barley  heads.  Agar  cultures  were 
incubated  at  the  following  temperatures:  5°,  14°,  18°,  24°,  28°,  32°, 
and  36°  C.  The  cultures  grown  at  5°  and  36°  did  not  produce  spores. 
The  barley  head  cultures  were  incubated  at  24°  C.  Fresh  barley 
leaves  were  taken  from  the  greenhouse,  placed  in  moist  chambers, 
inoculated  with  spores  of  the  same  culture,  and  incubated  at  24°.  The 
length  of  time  required  for  the  cultures  to  sporulate  at  the  different 
temperatures  varied  considerably;  those  at  24°,  28°,  and  32°  were 
ready  for  measurement  in  16  days,  while  those  at  14°  required  37  days. 


I 


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TECHNICAL  BULLETIN  17 


Spores  from  these  various  sources  were  then  measured  for  length. 
In  all  cases,  measurements  were  made  with  a  Bausch  and  Lomb  micro- 
scope, using  the  4  mm.  objective  and  an  eyepiece  micrometer  calibrated 
so  that  one  space  was  equal  to  3.4  microns.  It  was  observed  that  at 
an  extreme  temperature,  such  as  32°  C,  there  was  a  great  deal  of 
variation  in  length  and  a  large  number  of  measurements  would  be  re- 
quired to  obtain  a  normal  curve.  Data  were  therefore  recorded  for 
the  measurements  of  100,  300,  500,  and  1000  spores.  From  these  data 
the  mean  was  calculated  for  each  group  and  the  differences  were  com- 
pared in  relation  to  the  probable  errors,  according  to  the  methods  given 
by  Babcock  and  Clausen  (2).  These  data  are  summarized  in  Tables  I 
and  II.  For  100  spores  the  mean  was  found  to  be  62.30  ±  0.85  microns ; 
for  300  spores  57.44  ±  0.57  microns;  for  500  spores  59.66  ±  0.45 
microns;  and  for  1000  spores  59.07  ±  0.31  microns.  Their  accuracy 
can  be  seen  by  comparing  these  results  with  their  probable  errors.  In 
Table  II  the  comparison  of  the  means  for  100  and  300  spores  with 
the  means  of  each  of  the  other  three  groups,  shows  that  the  difference 
between  any  two  is  from  3  to  5  times  the  probable  errors  of  the  differ- 
ence. This  borders  on  the  verge  of  a  significant  difference,  so  that 
100  or  perhaps  even  300  spores  are  scarcely  enough  to  use  as  a  basis 
for  drawing  conclusions.  When  the  mean  for  500  spores  is  compared 
with  that  for  1000,  the  ratio  is  i  :i.  The  results  obtained  by  measuring 
1000  spores  are  only  very  slightly  more  accurate  than  those  obtained 
by  measuring  500  spores.  The  difference  is  certainly  not  great  enough 
to  necessitate  the  measurement  of  the  second  500  spores. 

TABLE  II 

SUMMARY  OF  COMPARISONS  BETWEEN  MEANS  AND  COEFFICIENTS  OF  VARIABILITY  FOR  LENGTH  OF 

SPORES  OF  Helminthosporium  sativum  OBTAINED  FROM   MEASURING  POPULATIONS 

OF  DIFFERENT  SIZE  (FROM  DATA  SUMMARIZED  IN  TABLE  I) 


Means 

Coefficients  of  variability 

Conditions 
compared 

Difference 

Difference  divided  by 
the  probable  error 
of  the  difference 

Difference 

Difference  divided  by 
the  probable  error 
of  the  difference 

No.  of  spores 

100  and    300 

4.86+1  .02 

5 

5-4i±i.73                              3 

100  and    500 

2.64  +  0.96 

3 

4.66+1.31 

4 

100  and  1000 

3.23±o.9i 

4 

4.  ii  ±1.24 

3 

300  and    500 

2.22  +  0.72 

3 

o.75±o.Q3 

i 

300  and  1000 

1.63  +  0.65 

3 

i  .30  +  0.85 
o.55±o.68 

2 

500  and  1000 

o.59±o.55 

i 

I 

PATHOGENICITY  OF  H.  SATIVUM 


ii 


These  comparisons  are  perhaps  brought  out  more  clearly  by  the 
curves  in  Figure  I,  in  which  the  data  obtained  from  measuring  the 
different  lots  of  spores  have  been  plotted  after  grouping  the  measure- 
ments into  10  micron  classes.  The  lowest  curve,  representing  100 
spores,  very  clearly  does  not  give  a  true  index  of  the  lower  extreme 
of  the  total  population.  This  explains  why  the  mean  obtained  from 
100  spores  is  too  high.  The  three  succeeding  curves  show  that,  as 
the  number  of  individuals  increases,  the  curve  gradually  approaches 


Fig.    i.      Types  of  Curves   Obtained   from   Measuring   the   Length   of    100,    300,    500,   and    1000 

Spores   of   Hclminthosporium   sativum    Produced   on    Potato    Dextrose 

Agar  at   32°    C. 


iz  TECHNICAL  BULLETIN  17 

a  normal  one.  In  general  contour  the  3OO-spore  curve  and  the  500- 
spore  curve  approach  the  looo-spore  curve,  altho  the  first  is  somewhat 
more  irregular.  The  slight  rise  at  the  lower  extreme  indicates  that 
the  short  spores  tend  to  group  themselves  about  a  mode  of  their  own. 
It  is  possible  that  improvement  in  the  method  of  sampling  might  in- 
crease the  accuracy  of  the  results  obtained  from  a  smaller  population. 
In  the  present  study  about  100  spores  were  measured  from  one  mount. 
The  spores  were  distributed  as  evenly  as  possible  in  the  drop  of  water 
and  each  spore  was  measured  in  passing  systematically  over  the  slide 
from  the  upper  left  to  the  lower  right  hand  corner.  An  attempt  was 
made  to  make  the  mount  so  that  two  or  three  spores  would  come  into 
the  field  at  once.  For  all  other  conditions,  500  spores  were  measured. 

Results  obtained  in  the  study  of  the  morphology  of  spores  de- 
veloped on  potato  dextrose  agar  at  various  temperatures  are  interesting. 
Table  III  shows  very  little  difference  in  the  means  of  spores  de- 
veloped at  1 8°  and  24°.  From  the  comparisons  in  Table  IV  it  is  seen 
that  these  differences  are  insignificant.  If,  however,  we  examine  the 
coefficients  of  variability,  we  find  that  there  is  a  significant  difference 
in  the  amount  of  relative  variation  in  the  length  of  spores.  This  fact 
is  very  clearly  brought  out  in  the  curves  in  Figure  2.  The  degree  of 
variation  is  not  increased  by  a  temperature  4  degrees  lower  (14°  C.)> 
but  the  mean  length  of  the  spores  is  slightly  increased.  This  may  be 
due  to  the  fact  that  at  a  lower  temperature  the  black  outer  wall  on 
the  spores  and  mycelium  is  laid  down  much  more  slowly,  so  that  the 
spores  have  a  longer  time  in  which  to  form.  This  is  further  sub- 
stantiated by  the  fact  that  at  32°  the  spores  are  very  much  shorter. 
The  amount  of  relative  variation  is  practically  the  same  as  at  the 
lower  extreme.  These  differences  in  length  of  spores  produced  at 
various  temperatures  are  graphically  represented  by  the  curves  in 
Figure  2. 

The  most  striking  difference  in  spore  morphology  was  obtained 
by  comparing  the  spores  produced  on  different  media.  As  the  fresh  leaf 
and  the  autoclaved  head  cultures  were  incubated  at  24°  C,  we  may 
compare  these  results  with  those  pbtained  from  the  agar  culture  at 
24°  C.  Comparing  first  the  spores  from  the  head  and  from  the  agar, 
we  find  that  the  former  are  slightly  longer.  The  amount  of  relative 
variation  in  the  two  is  practically  the  same.  On  the  fresh  leaves, 
however,  the  spores  are  very  much  longer  and  decidedly  more  uniform. 
These  differences  are  illustrated  in  the  curves  in  Figure  3. 


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14  TECHNICAL  BULLETIN  17 

TABLE  IV 

SUMMARY  OF  COMPARISONS  BETWEEN  MEANS  AND  COEFFICIENTS  OF  VARIABILITY  FOR  LENGTH  o* 

SPORES  OF  Helminthosporium  sativum  PRODUCED  UNDER  DIFFERENT  CONDITIONS 

FROM   DATA  SUMMARIZED  IN   TABLE  III. 


Means                                                  Coefficients  of  variability 

Conditions 
compared 

Difference 

Difference  divided  by  !                                  Difference  divided  by 
the  probable  error             Difference             the  probable  error 
of  the  difference                                              of  the  difference 

14°  C.  and  18°  C. 

2.3o±o.75 

3                             0.80 

+  0.86     !                        i 

14°  C.  and  24°  C. 

i  .57  +  0.66 

2                                           8.62 

±0.74       i                              12 

14°  C.  and  28°  C. 

n-34±o.6s 

17                             6.81 

±0.77                            9 

14°  C.  and  32°  C. 

7.66  +  0.  71 

II                                         2.  48  ±0.83       :                               3 

18°  C.  and  24°  C. 

0.73  +  0.64 

I                                         7.82  +  0.73                                   II 

18°  C.  and  28°  C. 

9.04  +  0.63 

14                             6.01  +o.  76     , 

18°  C.  and  32°  C. 

5.36  +  0.68 

8                             1.68  +  0.82                           2 

24°  C.  and  28°  C. 

9-77±o.5i 

19                             1.81 

±0.62                            3 

24°  C.  and  32°  C. 

6.o9±o.s8 

ii                             6.14 

+  0.69     i                       9 

28°  C.  and  32°  C 
Head  and  leaves 

6.32±o.£6 

ii                             7.44 

+  0.73                         10 

15.40  +  0.47 

33                                7-49 

+  0.47                          16 

Head  and  ajar 
24°  C. 

i  -99±o.53 

4                                0.04 

±0.57                            o 

Leaves  and  ag-r 
24°  C. 

i7-39±o.47 

37                               7-53 

±0.48                         1  6 

Leaves  and  agar 
28°  C. 

27  .  16  +  o  .  45 

60                              9.34 

±0.52                         18 

These  facts  show  that  in  a  single  spore  strain  of  a  Helnuntho- 
sporium  of  this  type,  marked  variations  may  be  found  in  the  length 
of  spores  developed  under  various  conditions.  Differences  in  spore 
measurements  by  various  authors  are  therefore  to  be  expected,  and 
very  fine  specific  or  varietal  differences  can  not  be  drawn  on,  the  basis 
of  spore  size  unless  a  large  number  of  carefully  controlled  compara- 
tive studies  have  been  made.  Seemingly,  the  original  spore  meas- 
urements given  by  Pammel,  King  and  Bakke  are  rather  large  (105 
to  130  microns).  Stevens  has  come  nearest  to  approaching  this  length 
with  a  maximum  of  122  microns.  The  same  author  states  that  the 
majority  of  his  spores  fall  within  80  to  90  microns.  By  examining 
Table  III  it  will  be  seen  that  the  majority  of  the  spores  developed 
upon  fresh  barley  leaves  in  a  moist  atmosphere  fall  within  the  80  and 
90  classes,  or  within  75.0  to  94.9  microns.  Out  of  4000  spores  meas- 
ured, only  43. were  longer  than  100  microns.  On  the  fresh  leaves  the 
longest  spore  measured  was  only  115.5  microns.  However,  the  opti- 
mum conditions  for  maximum  and  minimum  spore  length  have  not 


PATHOGEN rICITY  OF  H.  SATIVUM 


necessarily  been  obtained  in  these  studies.  On  the  basis  of  spore 
shape  and  similarity  with  the  organism  obtained  from  typical  spot 
blotch  lesions  and  the  ability  to  produce  spot  blotch  symptoms  on  barley, 
the  organism  undoubtedly  should  be  included  in  the  species  Helmin- 
th osporium  sativum  P.K.B. 


Z.-40- 


00. 


60 


20 


80 


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24-°  C 
2.8°  C 
32°  C 


!\ 


1-t 


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2O 


60 


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Lengfh   of  spores     (microns) 

Fig.    2.      Length   of   Spores   of   Helniinthosporinm   satii'itin    Produced   on    Potato    Dextrose   Agar 
.     at  Various  Temperatures 

The  shape  of  the  spores  was  found  to  be  more  or  less  the  same 
under  various  conditions.  At  24°  and  28°,  the  spores  tended  to  be 
fat,  spindle-shaped  to  broadly  elliptical,  sometimes  slightly  curved. 
At  32°  the  thickening  in  the  middle  was  less  evident,  and  they  tended 
to  be  more  uniform  in  diameter.  The  small  spores  were  globose  to 
ovate.  At  14°  the  longest  spores  were  mostly  narrowly  cylindrical. 
There  was  a  marked  tendency  for  the  thickened  portion  to  occur 
nearer  the  base  than  the  apex,  giving  the  spore  the  shape  of  a  slender 
flask. 


i6 


TECHNICAL  BULLETIN  17 


Throughout  the  culture  work,  bizarre  forms  frequently  appeared, 
especially  forked  spores  which  were  sometimes  almost  stellate.  Seven 
or  eight  such  single  spores  were  isolated  and  planted  on  agar  slants. 
In  each  case  normal  spores  were  produced  and  the  bizarre  type  occurred 
so  rarely  that  it  was  quite  impossible  to  isolate  another  single  spore  of 
the  same  shape  from  the  progeny. 

240 


O  20  4O  6O  &O 

Lengfh    of  <spore<5    (microns) 


too 


/2O 


Fig.  3.     Length  of  Spores  of  Helminthosporium  sattivum  Produced  on  Different 

Substrata  at  24°  C. 

The  solid  line  represents  spores  produced  on  autoclaved  ripe  barley  heads;  the  line  with 
short  dashes,  spores  produced  on  potato  dextrose  agar;  the  line  with  alternate  long  and  short 
dashes,  spores  produced  on  fresh  barley  leaves  in  a  moist  chamber. 

TEMPERATURE  RELATIONS 
GROWTH  OF  FUNGUS  ON  POTATO  DEXTROSE  AGAR 

In  determining  the  temperature  relations  of  H.  sativum,  the  first 
problem  studied  was  the  growth  of  the  fungus  in  pure  culture.  In  all, 
four  series  were  run  to  determine  the  range  of  growth,  the  same  gen- 
eral method  being  used  in  each.  Thirty  cubic  centimeters  of  potato 
dextrose  agar  were  poured  into  petri  dishes  10  centimeters  in  diameter. 


PATHOGENICITY  OF  H.  SATIVUM 


The  plates  were  inoculated  in  the  center  and  incubated  at  the  various 
temperatures.  Each  series  was  run  in  triplicate.  The  diameter  of  the 
colony  was  taken  as  the  index  of  growth.  In  some  cases  it  was  impos- 
sible to  control  the  temperature  within  several  degrees,  so  that  one  series 
can  not  be  checked  quantitatively  against  the  other.  Different  lots  of 
potato  dextrose  also  were  used  in  the  different  series.  The  results  of 
two  series  are  given  in  Table  V.  In  each  case,  the  size  of  the  colony 
represents  the  average  of  three  plates. 

TABLE  V 

EFFECT  OF  TEMPERATURE  ON   GROWTH  OF  Helminthosporium  sativum  on  Potato  Dextrose  Agar 


First  Series                                                                          Second  Series 
(Feb.  28-March  3,  1921)                                                      (Dec.  15-22,  1921) 

Temperature, 
degrees  C. 

Diameter  of  colony 
(after  9  days) 

Temperature, 
decrees  C. 

Diameter  of  colony 
(after  7  days) 

o-  2 
6-  8 
13-15 

mm. 
4 
*4 
28 

3-  6 
12-13 
15-18 

mm. 
9 
18 
30 

17-22 
21-24 

Si 

20-23 
27-28 

77 
By 

30-32 

36 

31-33 

35 

34-35 
40-42 

13 

0 

35-39 

4 

/C 

6 
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Temp  erature   (  °  Cen  tig  rade) 

O  5  /O  /S  2O 


35- 


Fig.   4.      Growth    of    Hclminthosporinm    sativum    on    Potato    Dextrose    Agar    in    Petri    Dishes 
The  curve  represents  the  diameter  of  the  colonies  at  the  end  of  seven  days. 

Data  obtained  in  the  second  series  are  shown  graphically  in  the 
curves  of  Figures  4  and  5.  Figure  4  shows  the  relative  growth  of 
the  fungus  at  the  various  temperatures  after  seven  days.  Figure  5 


i8 


TECHNICAL  BULLETIN  17 


shows  the  daily  rate  of  growth  at  each  of  the  temperatures  tested. 
Plate  I  shows  the  final  appearance  of  the  colonies  in  the  first  series, 
Plate  II  in  the  second. 

From  these  results  we  may  conclude  that  the  minimum  temperature 
for  the  growth  of  Helminthosporium  sativum  lies  near  0-2°  C.,  the 
maximum  temperature  between  35°  and  39°  C.  and  the  optimum  be- 
tween 24°  and  28°  C. 


28° C 


2^0c 


Fig.    5.      Daily    Rate   of    Growth    of   Helminthosporium    sastivum    on    Potato    Dextrose    Agar    in 

Petri  Dishes 


PATHOGENICITY  OF  H.  SATIVUM  ig 

SPORE  GERMINATION 

In  the  first  series  of  studies  to  determine  the  effect  of  temperature 
on  spore  germination,  hanging  drop  cultures  were  made  on  the  covers 
of  petri  dishes,  using  distilled  water  and  Czapek's  solution,  minus  the 
sugar,  as  media.  The  spores  were  taken  from  a  six-days-old  bean 
agar  culture.  Germination  counts  were  made  after  48  hours.  The 
results  are  given  in  Table  VI. 

TABLE  VI 

SPORE  GERMINATION  OF  H.  sativitm  IN  DISTILLED  WATER  AND  IN  CZAPEK'S  SOLUTION   AT 
VARIOUS  TEMPERATURES 


Tempera- 


Distilled water  PH  6.7 


Czapek's  solution  —  sugar  PH 


grees  C 

ISt 

drop 

-Mid     3rd 
drop    drop 

4th 
drop    Av. 

ISt 

drop 

2nd 

drop 

3rd 

drop 

4th 
drop 

Av. 

i 

V 

i—     i  — 

i  —     i  — 

20 

I  0 

15 

10 

14 

6 

10 

8        20 

13 

30 

50 

40 

53 

42 

14 

10 

20 

15 

55 

50 

62 

40 

52 

18 

40 

40        30 

37 

66 

50 

60 

59 

24 

68 

41      44 

58 

85 

86 

75 

75 

80 

30 

45 

65      48 

4i      50 

77 

70 

59 

80 

72 

34 

50 

60 

55 

36 

50 

25 

4- 

38 

42 

0 

0         0 

O         0 

0 

0 

0 

0 

0 

With  the  exception  of  the  results  obtained  at  34°  C.,  a  higher 
percentage  of  germination  was  obtained  in  Czapek's  solution  than  in 
distilled  water.  In  both  cases  the  optimum  occurred  at  -24°.  At  a 
temperature  of  i°  C.  14  per  cent  of  the  spores -germinated  in  Czapek's 
solution,  while  less  than  i  per  cent  germinated  in  distilled  water.  At 
42°  no  germination  occurred  in  either  case,  while  at  34°  the  germina- 
tion in  distilled  water  was  practically  the  same  as  at  30°,  but  in  Czapek's 
solution  a  marked  inhibition  occurred  at  the  higher  temperature. 

After  trying  various  methods  for  germinating  spores,  including 
hanging  drops  in  petri  dishes,  in  van  Tieghem  cells,  films  on  slides 
in  moist  chambers  and  in  moist  atmosphere,  the  most  satisfactory 
method  proved  to  be  floating  the  spores  on  the  surface  of  a  thin  layer 
of  a  liquid  medium  in  Syracuse  watch  glasses.  In  such  cultures  the 
spores  can  be  counted  directly  on  the  surface  under  the  low  power  of 
the  microscope. 

In  several  series,  through  the  different  temperature  ranges,  con- 
sistently high  percentages  of  germination  were  obtained  at  the  extreme 
temperatures  when  water  which  had  been  redistilled  over  glass  was 


2O 


TECHNICAL  BULLETIN  17 


used.  Fluctuations  occurred  in  the  different  cups  at  any  one  tempera- 
ture. The  results  given  in  Table  VII  are  typical.  The  percentages 
given  in  the  table  represent  the  average  of  several  counts. 

TABLE   VII 
SPORE  GERMINATION  OF  H.  sativum  IN  REDISTILLED  WATER  AT  VARIOUS  TEMPERATURES 


Temperature,  degrees  C. 

Average  percentage  of  germination 

Spores  floating  on 
surface  of  water 

Spores  lying  on 
bottom  of  cup 

5-5-  6.5 

87 

76 

11.5-12.0 

93 

89 

17.0-19.0 

73 

57 

21.0-24.0 

62 

61 

28.0-29.5 

70 

66 

30.5-32.0 

83 

46 

34.0-35.0 

65 

63 

38.0-39.0 

65 

In  a  second  series,  using  hanging  drops  in  petri  dishes,  67  per  cent 
of  the  spores  germinated  at  6°,  54  per  cent  at  12°,  79  per  cent  at  18°, 
91  per  cent  at  22°,  72  per  cent  at  28°,  91  per  cent  at  29-30°,  80  per 
cent  at  32°,  82  per  cent  at  35°,  and  87  per  cent  at  39°. 

In  a  third  and  fourth  series  in  watch  crystals,  the  spores  were  not 
counted  but  the  germination  was  indicated  as  poor,  moderate,  and 
good. .  After  24  hours  incubation,  in  these  series,  the  germination 
was  poor  at  6°,  moderate  to  good  at  12°,  18°,  and  22°,  and  good  at 
the  higher  temperatures.  By  count,  89  per  cent  of  the  spores  germinated 
at  39°.  At  the  end  of  48  hours  the  germination  was  good  at  6°. 

From  these  results  with  redistilled  water  it  is  difficult  to  detect 
any  quantitative  effect  of  temperature  on  the  number  of  spores  which 
germinate.  Even  at  i°  C.  a  small  number  of  spores  will  germinate. 
This,  however,  is  probably  very  near  the  lower  limit.  At  the  lower 
temperatures,  i°,  6°,  and  12°,  pieces  of  mycelium  in  the  cultures 
always  germinated  much  more  readily  and  sent  out  longer  tubes  than 
did  the  spores.  At  40-42°  no  germination  occurred  in  the  first  series 
for  which  the  results  are  given.  In  later  series,  however,  high  germi- 
nation sometimes  occurred  at  38-39°.  Comparing  these  results  with 
the  data  presented  in  Table  V,  we  find  35-39°  to  be  the  maximum 
temperature  for  the  growth  of  the  mycelium  on  potato  dextrose.  At 
temperatures  as  high  as  35°  and  39°  the  germ  tubes  appeared  very 
quickly,  but  were  always  short  and  did  not  increase  much  in  length 
after  two  or  three  days.  On  the  other  hand,  at  22°,  28°,  and  32°, 


PATHOGENICITY  OF  H.  SATIVUM  21 

the  tubes  formed  such  a  mat  of  mycelium  by  the  end  of  24  hours  that 
it  was  often  difficult  to  determine  the  percenteige  of  germination.  At 
the  lower  temperatures  a  longer  time  was  required  for  the  germ  tubes 
to  appear,  and  they  increased  in  length  very  slowly. 

In  redistilled  water,  therefore,  spores  of  H.  sativum  germinate 
about  equally  well  at  temperatures  from  6°  to  39°  C.  No  very 
definite  optimum  temperature  for  germination  is  apparent.  The  char- 
acter of  the  germ  tubes  and  the  length  of  time  in  which  they  appear, 
however,  would  indicate  that  an  optimum  temperature  lies  between 
22°  C.  and  32°  C.  From  these  results  it  would  seem  that  for  the 
above-ground  parts  of  the  host,  temperature  is  not  a  limiting  factor 
in  infection  so  far  as  spore  germination  is  concerned. 

From  the  data  in  Table  VII  it  will  be  seen  that  in  most  cases  the 
percentage  of  germination  of  the  submerged  spores  is  only  slightly  less 
than  that  of  those  on  the  surface.  In  all  cases,  however,  the  germ 
tubes  produced  under  water  were  very  short  and  abnormally  branched 
in  comparison  with  the  long  straight  tubes  produced  on  the  surface. 

The  germ  tube  first  appears  as  a  hyaline  tip  at  the  apex  of  the 
spore.  It  is  difficult  to  determine  whether  the  tube  breaks  through 
the  wall  or  emerges  through  a  pore.  After  the  tube  has  increased  in 
size,  the  delicate  exospore  is  split,  sometimes  for  a  third  of  the  length 
of  the  spore.  A  second  tube  soon  appears  at  the  base  of  the  spore, 
just  to  one  side  of  the  scar  where  the  spore  was  attached  to  the!  sporo- 
phore.  The  connection  between  the  two  tubes  is  continuous  through 
the  spore,  showing  the  false  nature  of  the  septation  in  the  endospore. 
The  endospore  is  frequently  drawn  away  from  the  exospore  and  forms 
a  constricted  tube  through  the  latter.  Two  germ  tubes  are  not  always 
formed  from  each  spore.  In  one  lot  of  spores  germinated  in  redis- 
tilled water  at  22°  C.,  it  was  found  that  52  per  cent  of  the  spores  pos- 
sessed germ  tubes  at  both  ends  and  39  per  cent  at  only  one  end.  Nine 
per  cent  of  the  spores  did  not  germinate.  Very  rarely  lateral  tubes 
are  found.  One  spore  was  observed  with  a  lateral  germ  tube  from 
each  of  five  adjacent  cells  at  one  end  of  a  ten-celled  spore.  In  a  few 
other  cases,  one  or  two  lateral  tubes  were  observed,  usually  arising 
from  central  cells.  Fusions  between  germ  tubes  are  very  common. 

EFFECT    OF   HYDROGEN-ION    CONCENTRATION    AND    TEMPERA- 
TURE ON   SPORE  GERMINATION 

As  so  many  spores  always  germinated  in  redistilled  water  at  the 
various  temperatures  which  permitted  germination  at  all,  the  effect  of 
hydrogen-ion  concentration  on  germination  was  studied.  Culture  solu- 
tions, based  on  Clark  and  Lub's  (4)  titration  curve  for  ortho-phosphoric 
acid,  were  made  by  adding  varying  quantities  of  n/5  KOH  to  50  cc. 


22 


TECHNICAL  BULLETIN  17 


n/5  H3  PO4  to  give  a  series  of  hydrogen-ion  concentrations  ranging 
from  pH  2.4  to  pH  12.  The  pH  value  of  the  solutions  was  deter- 
mined colorimetrically,  except  for  the  highest  three  alkaline  solutions 
for  which  the  theoretical  value  according  to  the  curve  is  given.  Spores 
from  a  seven-weeks-old  barley  head  culture  were  dusted  over  the 
surface  of  the  solutions  in  Syracuse  watch  glasses.  The  percentage 
of  germination  was  determined  after  18  hours  and  after  36  hours. 
Similar  series  were  run  in  triplicate  at  19°,  24°,  and  32°  C.  The 
results  are  given  in  Table  VIII. 

TABLE  VIII 

SPORE  GERMINATION  OF  H.   sativum  IN   H3  PO4 — KOH   SOLUTIONS  OF  VARIOUS   HYDROGEN-ION 
CONCENTRATIONS  AT  DIFFERENT  TEMPERATURES 


PH 


Hours 


19°  C. 


Germination 
24°  C. 


32°  C. 


i 

2 

3 

Av. 

i 

2 

3 

Av. 

t 

2 

3 

Av. 

2.4 

18 

0 

0 

0 

0 

0 

o 

0 

0 

o 

0 

0 

0 

36 

3 

4 

i 

3 

o 

o 

o 

o 

0 

2 

o 

1 

3-4 

18 

2. 

o 

4 

2 

5 

6 

0 

4 

8 

10 

10 

9 

36 

2 

4 

7 

4 

4 

o 

2 

2 

10 

13 

7 

10 

4-4 

18 

IS 

17 

14 

15 

40 

36 

28 

35 

• 

44 

44 

28 

39 

36 

22 

19 

14 

18 

4i 

33 

16* 

30 

3i 

53 

27 

38 

5-2 

18 

27 

28 

25 

27 

55 

38 

46 

46 

40 

42 

54 

44 

36 

28 

33 

25 

29 

52 

39 

52 

48 

44 

4i 

52 

44 

6.4 

18 

31 

25 

40 

33 

So 

59 

57 

55 

47 

76 

52 

44 

36 

32 

33 

34 

33 

56 

64 

62. 

61 

4i 

66 

55 

54 

7-o 

18 

40 

42 

60 

47 

7i 

7i 

79 

74 

86 

76 

72 

76 

36 

36 

45 

53 

45 

68 

65 

84 

72 

65 

74 

60 

66 

7-4 

18 

30 

39 

68 

46 

81 

75 

72 

76 

78 

80 

84 

8c 

36 

34 

60 

67 

54 

85 

83 

73 

80 

72 

83 

87 

81 

7-8 

18 

55 

35 

36 

42 

83 

78 

80 

80 

88 

90 

94 

9i 

36 

30 

34 

35 

33 

83 

90 

90 

88 

97 

94 

92 

94 

8.o 

ii 

35 

24 

29 

29 

72 

62 

68 

67 

72, 

65 

74 

70 

36 

35 

22 

38 

32 

76 

68 

82 

75 

75 

79 

78 

77 

8.2 

18 

21 

18 

18 

19 

60 

43 

50 

Si 

70 

80 

90 

80 

36 

19 

24 

19 

21 

72 

6.1 

65 

66 

75 

85 

84 

81 

9.2 

18 

25 

40 

34 

33 

62 

58 

63 

61 

87 

82 

87 

84 

36" 

36 

47 

35 

39 

85 

70 

60 

72 

89 

90 

95 

1  1.4* 

18 

22 

22 

20 

21 

33 

40 

32 

35 

84 

17 

82 

~sT 

36 

40 

35 

36 

37 

73 

74 

82 

76 

86 

93 

90 

89 

1  1.  8* 

18 

12 

18 

12 

14 

0 

0 

8 

3 

40 

26 

39 

35 

36 

26 

34 

37 

32 

10 

0 

20 

TO 

30 

69 

48 

39 

12.0* 

18 

0 

o 

i 

o 

0 

o 

0 

O 

o 

i 

o 

o 

36 

2 

14 

i 

6 

o 

22 

0 

7 

9 

35 

o 

15 

*  Theoretical  value  according  to  Clark  and  Lub's  titration  curve  for  ortho-phosphoric 


acid. 


PATHOGENICITY  OF  H.  SATIVUM 


After  an  incubation  of  18  hours  at  19°  C.  no  germination  was 
obtained  at  a  hydrogen-ion  concentration  of  pH  2.4.  Very  slight 
germination  occurred  at  pH  3.4 ;  while  at  pH  4.4  the  germination 
showed  a  marked  increase,  rising  steadily  until  a  hydrogen-ion  concen- 
tration of  pH  7  was  reached.  From  this  point  a  gradual  decrease  oc- 
curred, reaching  the  lowest  point  at  pH  8.2.  At  pH  9.2  there  was  a 
second  rise  followed  by  a  gradual  falling  off,  until  at  pH  12  no  germi- 
nation occurred.  After  incubating  for  18  hours  longer  there  was 
scarcely  any  change  in  the  amount  of  germination  on  the  acid  side. 
There  was  a  slight  increase  on  the  alkaline  side. 

At  the  higher  temperatures  the  results  were  very  much  the  same 
except  that  the  percentage  of  germination  was  increased  and  the  point 
of  maximum  germination  was  shifted  slightly  to  the  alkaline  side. 
At  both  24°  and  32°  the  optimum  germination  occurred  on  the  alkaline 
side  of  neutrality  at  a  hydrogen-ion  concentration  of  pH  7.8.  A  much 
greater  increase  in  germination  occurred  at  the  higher  temperatures 
on  the  alkaline  side  than  on  the  acid.  The  average  germination  after 
1 8  hours  incubation  at  the  different  temperatures  is  represented  by 
the  curves  in  Figure  6. 


32*  C 
24-°  C 


Hydrogen    ion    concenrrahon   -  p  // 

3  -?  5  6  7  B  9  SO  It  l£ 

Fig.  6.     Percentage  Germination  of  Spores  of  Helminthosporium  sativnm  in  Phosphoric  Acid — 
Potassium    Hydroxide    Solutions    of    Various    Hydrogen-Ion    Concentrations 

Webb  (14)  germinated  spores  of  Aspcrgillus  nigcr,  Pcnicillium 
cyclopium,  Fusarium  sp.,  Botrytis  cinerea,  and  Lcnzites  saepiaria  in 
n/5  mannite  solutions  in  which  the  hydrogen-ion  concentrations  were 
adjusted  by  the  use  of  H3  PO4  and  NaOH  according  to  Clark  and 
Lub's  titration  curve  for  ortho-phosphoric  acid.  The  results  obtained 
with  Fusarium  sp.  are  the  only  ones  comparable  with  those  obtained 
with  H.  sativum  in  the  wideness  of  the  range  of  hydrogen-ion  con- 


TECHNICAL  BULLETIN  17 


centration  which  permits  spore  germination.  It  may  be  pointed  out 
that  both  Fusarium  and  Helminthosporium  are  chiefly  soil  organisms. 
Among  the  organisms  that  Webb  studied,  only  Fusarium  responded 
favorably  to  an  alkaline  medium.  Maximum  germination  occurred  at 
hydrogen-ion  concentrations  of  pH  2.8  and  pH  7.4.  From  pH  6.2 
a  steady  increase  in  germination  occurred  with  the  increase  in  hydrogen- 
ion  concentration  up  to  pH  2.8.  From  the  same  point,  a  steady  increase 
in  germination  also  occurred  with  the  decrease  in  hydrogen-ion  con- 
centration and  practically  the  same  maximum  was  reached  at  a  con- 
centation  of  pH  7.4.  Examining  the  data  of  H.  sativum  again,  there 
is  a  steady  decrease  in  germination  from  the  neutral  point  with  the 
increase  in  hydrogen  ions  up  to  a  concentration  of  pH  2.4,  where  no 
germination  occurred  during  18  hours  and  only  very  slight  germina- 
tion during  36  hours.  However,  the  usual  bimodal  curve  is  obtained, 
but,  in  this  case,  both  maxima  occur  on  the  alkaline  side  at  hydrogen-ion 
concentrations  of  pH  7.8  and  pH  9.2.  With  H.  sativum,  germination 
occurred  chiefly  in  the  alkaline  solutions. 

A  series  of  spore  germination  tests  was  also  made  in  Czapek's 
solution  minus  the  sugar,  with  various  hydrogen-ion  concentrations 
ranging  from  pH  2.6  to  pH  9.8.  The  results  are  represented  by  the 
curve  in  Figure  7.  In  this  case  also  the  bimodal  curve  was  obtained. 
The  first  maximum,  however,  occurred  on  the  acid  side  of  neutrality  at  a 
hydrogen-ion  concentration  of  pH  6.  The  second  was  on  the  alkaline 
side  at  a  concentration  of  pH  8. 


•IOO 


80 


3*56789  tC 

Hydrogen  ion   concentration  •  p  ti 

Fig.  7.     Percentage  Germination  of  Spores  of  Helminthosporium  sativum  in  Czapek's 
Solution    Minus  the   Sugar  at   Various   Hydrogen-Ion   Concentrations 


PATHOGENICITY  OF  H.  SATIVUM  25 

While  the  germination  of  //.  sativum  spores  in  these  solutions  is 
not  necessarily  the  same  as  in  a  soil  solution,  certain  general  relation- 
ships may  be  pointed  out.  The  spores  will  germinate  through  a  wide 
range  of  hydrogen-ion  concentration.  Optimum  germination  occurs 
near  the  neutral  point  or  on  the  alkaline  side.  The  spores  will  tolerate 
high  degrees  of  alkalinity.  Germination  studies  in  solutions  more 
nearly  approximating  soil  solutions  are  still  desirable  from  the  stand- 
point of  a  closer  analysis  of  the  development  of  the  disease. 

INFECTION 

Marquis  wheat  and  Lion  barley  were  grown  under  sterile  conditions 
in  test  tubes  containing  white  sand.  When  the  seedlings  were  about 
an  inch  high,  the  coleoptile  was  inoculated  with  a  suspension  of  spores 
and  incubated  at  various  temperatures.  At  22°,  25°,  and  30°  C.  char- 
acteristic minute  brown  lesions  were  visible  after  18  hours.  At  the 
end  of  five  days  no  infection  had  occurred  at  6°  on  the  barley ;  very 
light  infection  was  evident  on  the  wheat.  Light  infection  also  occurred 
on  both  wheat  and  barley  at  14°  and  34°,  and  on  wheat  at  30°.  Mod- 
erate to  heavy  infection  occurred  on  both  hosts  at  22°  and  25°,  and 
also  on  the  barley  at  30°.  In  these  cases,  the  typical  basal  browning 
characteristic  of  the  seedling  blight  occurred.  This  was  as  far  as  it 
was  possible  to  follow  the  disease  under  these  conditions.  The  results 
indicate  that  infection  will  take  place  to  some  extent  through  a  rather 
wide  range  of  temperature  from  6°  to  34°  C.  but  that  for  the  severe 
development  of  the  disease  the  range  is  narrower,  probably  22°  to 
30°.  To  some  extent  moisture,  as  well  as  temperature,  was  the  limit- 
ing factor  at  the  extremes. 

F.  L.  Stevens  (13)  reports  that,  "In  an  adaptation  of  the  rag-doll 
seed  tester,  which  allows  the  use  of  seedlings  under  aseptic  conditions 
and  variations  of  moisture  and  temperature  as  desired,  inoculation  by 
spores  of  Helminthosporium  upon  the  uninjured  sheath  was  followed 
within  24  hours  by  entrance  of  the  mycelium  into  the  host  cells,  and 
within  48  hours  by  a  browned,  diseased  spot  visible  to  the  naked  eye. 
Subsequently,  when  conditions  favored,  the  mycelium  invaded  the  inner- 
most leaves  and  caused  general  rotting  and  death.  When  inoculated 
upon  the  roots,  there  was  general  invasion  of  the  cortex  with  very  slight 
discoloration."  Stevens  does  not  report  under  what  conditions  of  tem- 
perature and  moisture  the  disease  developed  best. 

An  attempt  was  made  to  arrive  at  the  temperature  relations  gov- 
erning leaf  infection  by  inoculating  fresh  excised  leaves  with  spores 
of  H.  sativum,  placing  them  in  moist  chambers  and  incubating  them  at 
various  temperatures.  After  incubating  for  72  hours  at  6°  C.,  both 


26  TECHNICAL  BULLETIN  17 

inoculated  and  uninoculated  check  leaves  were  dark  green,  turgid,  and 
normal  in  appearance.  No  signs  of  infection  were  apparent.  Micro- 
scopic examination  showed  that  many  spores  had  germinated  but  so 
far  as  could  be  detected  from  free  hand  sections,  the  germ  tubes  had 
not  penetrated.  After  the  same  incubation  period,  at  12°  C,  very  small 
blue-green  water-soaked  areas  were  visible  at  the  points  of  inoculation. 
The  remainder  of  the  leaf  tissue  and  the  uninoculated  check  leaves 
were  still  green  and  normal  in  appearance.  These  water-soaked  areas 
were  not  yet  visible  at  the  end  of  48  hours'  incubation.  At  18°  C.,  by 
the  end  of  the  third  day,  there  were  green  water-soaked  areas  on 
which  conidiophores  were  beginning  to  appear  on  the  inoculated  leaves. 
The  tissue  of  the  leaves  was  still  firm  and  the  cells  were  turgid.  While 
the  infected  areas  retained  a  dark  blue-green  color,  the  rest  of  the 
leaf  was  yellow.  The  uninoculated  check  leaves  were  light  green  to 
yellow  in  color.  After  72  hours'  incubation  at  23°,  27°,  and  30°  C., 
there  were  large  dark  green  blotches  of  infected  tissue  covered  by  a 
velvety  mass  of  conidiophores.  The  leaf  tissue  was  beginning  to  soften 
and  the  check  leaves  and  non-infected  areas  were  yellow.  In  the  in- 
fected areas  the  cells  were  beginning  to  disintegrate,  but  the  chloroplasts 
were  still  green.  At  34°  C.  small  water-soaked  areas,  3  or  4  mm.  in 
diameter,  were  apparent  after  36  hours'  incubation.  At  this  time  the 
border  was  beginning  to  turn  brown.  By  the  third  day,  there  were  small, 
brown,  definite  leaf  spots,  similar  to  the  normal  lesions  produced  on 
leaves  in  the  greenhouse  and  in  the  field.  The  remainder  of  the  leaf 
tissue  and  the  check  leaves  were  yellow. 

Under  the  conditions  just  described,  there  was  always  an  abundance 
of  moisture,  so  that  the  difference  in  reaction  must  have  been  clue  to  the 
influence  of  temperature  on  host  and  fungus.  During  the  first  36 
hours  the  results  were  probably  more  or  less  comparable  to  results 
obtained  in  growing  leaves  attached  to  the  plant;  during  the  second 
36  hours,  at  some  temperatures  at  least,  the  relationship  was  probably 
saprophytic.  The  most  that  can  be  claimed  for  results  obtained  in  this 
way  is  that  they  are  only  indicative  of  what  may  happen  on  growing 
plants. 

The  results  obtained  from  these  experiments  would  indicate  that 
at  temperatures  of  from  18°  to  30°  C.,  penetration  into  the  leaf  will 
take  place  about  equally  well  in  the  presence  of  sufficient  moisture. 
Below  24°  the  spots  increase  in  size  more  slowly,  above  24°  more 
rapidly.  At  12°  a  much  longer  incubation  period  is  necessary  for  the 
development  of  water-soaked  areas  than  at  higher  temperatures.  At 
6°  no  visible  infection  was  obtained.  At  a  temperature  as  high  as  34°, 
on  the  other  hand,  the  development  of  the  spots  and  the  browning  of 


PATHOGENICITY  OF  H.  SATIVUM  27 

the  host  tissue  occurred  so  rapidly  that  further  development  of  the 
fungus  was  checked. 

While  no  control  experiments  were  made  with  soil  or  leaf  infection 
on  growing  plants,  results  obtained  in  the  greenhouse  agreed  in  general 
with  those  obtained  on  the  temperature  relations  of  the  fungus.  When 
the  average  temperature  was  between  75°  and  85°  F.,  much  better  infec- 
tion was  obtained  than  when  the  average  was  lower.  Better  results 
were  obtained  on  an  inner  bench  over  the  steam  pipes  than  on  an  outer 
bench  next  the  outside  wall  on  the  west  end  of  the  house  where  it 
was  always  cool,  and  vigorous  plants  developed  in  spite  of  heavy  Foil 
inoculation. 

These  results  also  agree  with  those  reported  by  McKinney  (8). 
He  says,  "Controlled  soil  temperature  experiments,  conducted  in  the 
'Wisconsin  temperature  tanks,'  and  field  experiments  show  that  seedling 
infection  hi  both  spring  and  winter  wheat  and  in  spring  barley  is  great- 
est at  relatively  high  temperatures.  The  optimum  temperature  appar- 
ently lies  between  26°  and  28°  C.  This  is  very  near  the  optimum  rate 
of  growth  of  H.  satiznim  in  pure  culture." 

INFLUENCE  OF  TYPE  OF  SOIL 

The  statement  has  already  been  made  that  particularly  severe  infec- 
tions of  Helminthosponuni  foot-  and  root-rots  were  observed  during 
the  summer  of  1920  on  sandy  soils  and  on  peat  soils  in  certain  localities 
in  Minnesota.  Consequently  one  of  the  first  tests  undertaken  was  a 
study  of  the  development  of  the  disease  in  different  types  of  inoculated 
soil  in  order  to  gain,  if  possible,  an  insight  into  the  individual  factors 
which  might  be  influencing  the  situation. 

A  heavy  loam,  a  sandy  loam,  a  sand,  and  a  peat  soil  were  selected 
for  use.  The  heavy  loam  was  a  black  dirt  used  without  modification ; 
the  sandy  loam  was  obtained  by  mixing  two  parts  of  the  heavy  loam 
with  one- part  of  quartz  sand;  and  the  sandy  soil  by  mixing  one  part 
of  the  heavy  loam  with  two  parts  of  coarse  sand.  All  this  soil  was 
passed  through  a  5-millimeter  mesh  screen  before  being  packed  into 
the  pots.  The  peat  was  a  high-lime  peat  obtained  from  Anoka  County 
through  the  Division  of  Soils,  and  fertilized  according  to  directions 
with  acid  phosphate  and  potassium  chloride  to  secure  maximum  yield 
from  this  particular  type  of  soil  (i). 

Small  pots  of  steam-sterilized  soil  were  planted  with  Marquis  wheat 
and  Lion  barley.  After  the  seeds  were  planted,  the  soil  was  watered 
several  times  with  a  heavy  suspension  of  Helminthosporium  spores. 


28 


TECHNICAL  BULLETIN  17 


When  the  plants  became  crowded  in  the  small  pots,  they  were  trans- 
planted to  larger  pots  containing  sterilized  soil  which  had  been  inocu- 
lated in  the  same  way.  In  these  pots,  the  plants  were  grown  to 
maturity. 

There  was  no  very  serious  seedling  blight  in  any  of  the  pots.  The 
coleoptiles  of  most  of  the  plants  were  darkened,  and  lesions  were  formed 
on  the  first  leaves.  The  seedlings  in  the  inoculated  soils  were  not 
noticably  smaller  than  those  in  the  uninoculated,  sterilized,  check 
soils.  When  about  six  weeks  old  the  height  of  the  plants  was  meas- 
ured in  order  to  determine  the  effect  of  the  disease  on  growth.  The 
results  are  given  in  Table  IX.  In  each  case  the  table  gives  the  average 
height  of  30  to  40  plants. 

Any  differences  in  height  of  the  plants  in  the  different  soils  in 
either  the  uninoculated  or  inoculated  series  may  be  considered  due 
to  the  influence  of  the  soil  in  which  they  grew.  As  will  be  seen  from 
Table  IX,  the  differences  between  the  plants  in  the  different  types 
of  soil  in  the  inoculated  series,  altho  small,  agree  fairly  well  with 
similar  differences  in  the  uninoculated  series.  The  differences  between 
check  plants  and  inoculated  plants  in  the  same  type  of  soil  may  be 
considered  to  be  the  result  of.  the  disease.  A  comparison  of  the  dif- 
ferences between  plants  in  inoculated  and  uninoculated  soils  of  the 
different  types  w^ill  give  an  index  of  the  influence  of  the  soil  type  on 
the  development  of  the  disease. 

TABLE  IX 

AVERAGE  HEIGHT  OF  WHEAT  AND  BARLEY  PLANTS  GROWN   IN   INOCULATED  AND 
UNINOCULATED  SOILS  OF  VARIOUS  TYPES 


Type  of  soil 

Marquis  wheat 

Lion  barley 

Inoculated 

Check 

Inoculated 

Check 

Heavy    loam 

cm. 

22.8 

cm. 
30.2 

cm. 
24-5 

cm. 
27.6 

Sandy  loam 

21.  0 

32.3 

29.0 

3i-i 

Sand 

23-0* 

33-6* 

25-3 

28.3 

Peat 

29-4* 

4i.7* 

29.4 

33-6 

*  Plants  were  measured  two   days  later  than  those  in  the  heavy  loam   and  in  the  sandy 
loam,  and»so  can  not  be  compared  with  these. 

Judging  by  the  height  of  the  plants  at  this  stage,  the  barley  de- 
veloped about  equally  well  in  the  heavy  loam  and  in  the  sand,  the 
difference  in  the  average  height  being  8  millimeters  in  the  inoculated 
series  and  7  millimeters  in  the  check  series.  The  increases  over  this 
amount  were  about  equal  in  the  sandy  loam  and  in  the  peat,  the  advan- 
tage being  slightly  in  favor  of  the  latter.  The  difference  between  {he 
height  of  plants  in  inoculated  and  uninoculated  soils  was  practically  the 


PATHOGENICITY  OF  H.  SATIVUM  29 

same  in  the  heavy  loam  and  in  the  sand.  This  would  indicate  that  these 
two  types  of  soil  had  practically  an  equal  influence  on  the  development 
of  the  disease. 

The  difference  was  less  pronounced  in  the  sandy  loam,  showing  that 
here  the  disease  had  least  influence  on  the  size  of  the  plants.  The  great- 
est difference  in  height  was  in  the  peat  soil,  indicating  that  here  the 
disease  had  most  influence  on  the  growth  of  the  plant.  From  these 
results  it  is  apparent  that  root-rot  of  barley  produced  the  greatest  effect 
on  the  host  in  the  peat,  a  less  marked  effect  in  the  sand  and  heavy  loam, 
and  the  least  effect  in  the  sandy  loam.  On  the  whole,  the  differences 
were  very  small.  The  further  development  of  the  disease  on  the  barley 
plants  was  not  followed. 

The  conclusions  to  be  drawn  from  the  height  of  the  wheat  plants  at 
this  stage  must  be  derived  from  comparisons  between  the  differences  in 
the  height  of  diseased  and  check  plants  in  the  same  type  of  soil.  It  is 
obvious  that  the  effect  of  the  disease  is  much  more  marked  on  the  wheat 
than  on  the  barley.  The  least  effect  of  the  disease  on  the  growth  of  the 
plants  was  obtained  in  the  heavy  loam.  There  was  practically  an  equal 
increase  in  effect  in  the  other  three  types  of  soil. 

The  wheat  was  then  transplanted  to  larger  pots  of  inoculated  soil. 
In  each  case,  the  most  severely  diseased  plants  were  transferred.  After 
transplanting,  the  check  plants  grew  much  faster  than  the  diseased 
plants,  and  headed  several  days  earlier.  Plate  III  shows  the  compara- 
tive vigor  and  size  of  the  plants  in  the  different  types  of  soil  at  maturity. 
Final  observations  were  made  on  the  Marquis  wheat  just  before  the 
heads  began  to  turn  yellow.  The  plants  were  removed  from  the  soil, 
carefully  washed,  and  examined  for  foot-  and  root-rot. 

In  the  heavy  loam  soil,  both  diseased  and  check  plants  averaged 
3.5  culms  per  plant.  While  the  severity  of  infection,  measured  by  the 
degree  of  browning  at  the  base  of  the  plant,  was  moderate,  there  was 
very  little  difference  in  the  extent  of  the  root  systems.  The  check 
plants  headed  four  days  earlier  than  the  diseased  plants  and  were  con- 
siderably more  vigorous.  A  slight  browning  occurred  at  the  base  of 
most  of  the  mature  check  plants  which  resembled  slightly  a  light  infec- 
tion by  Helminthosporium.  The  lesions,  however,  were  less  definite 
and  no  organism  was  obtained  from  tissue  cultures.  H.  sativnm  was 
isolated  from  the  base  of  diseased  plants. 

In  the  sandy  loam,  the  average  number  of  culms  on  each  diseased 
plant  was  3,  on  each  check  plant  2.5.  The  basal  infection  was  moderate. 
There  was  little  difference  in  the  root  systems. 

In  sand  the  average  number  of  culms  on  each  diseased  plant  was  3, 
on  each  check  plant  2.  The  infection  at  the  base  of  the  diseased  plants 


30  TECHNICAL  BULLETIN  17 

ranged  from  moderate  to  heavy  minus.  The  root  systems  of  the  dis- 
eased plants  were  considerably  less  extensive,  brown  lesions  were 
numerous,  and  the  roots  were  very  easily  broken.  The  contrast  between 
diseased  and  check  plants  was  greatest  in  this  type  of  soil. 

In  the  peat  soil  the  average  number  of  culms  on  inoculated  plants 
was  2.7,  on  uninoculated  2.6.  Basal  infection  was  light  'to  moderate. 
There  was  very  little  difference  in  general  appearance  of  the  plants 
grown  in  inoculated  soil  and  in  ^uninoculated  soil.  The  best  plants  in 
both  series  were  obtained  in  the  peat  soil. 

Under  the  conditions  studied,  the  root-rot  inhibited  the  growth  of 
Lion  barley  most,  during  the  first  six  weeks,  in  the  peat  soil.  The 
effect  of  the  disease  was  less  evident  in  the  heavy  loam  and  the  sand, 
and  least  evident  in  the  sandy  loam.  During  the  same  period,  the 
growth  of  Marquis  wheat  was  least  inhibited  in  the  heavy  loam.  The 
effect  of  the  disease  on  the  growth  of  the  plants  was  markedly  in- 
creased, and  to  practically  the  same  extent,  in  the  other  three  types  of 
soil.  By  the  time  of  maturity,  however,  the  disease  had  developed 
much  more  severely  in  the  sand,  as  evidenced  by  the  smaller  size  of  the 
plants,  their  decreased  vigor,  the  amount  of  basal  browning,  and  the 
breaking  down  of  the  root  system.  The  effect  of  the  disease  was  almost 
as  severe  in  the  heavy  loam.  In  both  the  sandy  loam  and  the  peat 
there  was  only  a  very  slight  difference  between  the  plants  grown  in 
inoculated  and  uninoculated  soil. 

In  analyzing  the  factors  involved  in  these  various  soils,  it  may  be 
pointed  out  that  in  the  loam  soils,  in  addition  to  the  change  in  physical 
texture  brought  about  by  adding  increasing  quantities  of  sand  to  the 
original  heavy  loam,  there  has  been  a  dilution  of  the  mineral  nutrients 
of  the  host,  a  decrease  in  the  water-holding  capacity,  a  decrease  in  the 
amount  of  organic  matter  in  the  soil,  and  an  increase  in  the  amount  of 
soil  aeration.  All  these  factors  may  be  assumed  to  have  an  influence  on 
both  the  host  and  the  pathogene.  On  the  other  hand,  in  the  peat  soil, 
we  have  a  high  organic  content,  a  high  water-holding  capacity,  and  an 
optimum  of  mineral  nutrients  for  the  host.  The  abundant  moisture 
and  high  organic  content  of  the  peat  soil  should  seemingly  be  conducive 
to  extensive  saprophytic  growth  of  Helminthosporium,  thus  greatly  in- 
creasing the  amount  of  inoculum  and  the  chance  for  infection  of  the 
growing  host.  This  tendency,  however,  seems  to  be  counterbalanced  by 
the  optimum  conditions  offered  for  the  growth  of  the  host.  On  the 
other  hand,  the  greater  severity  of  the  disease  in  sand  and  heavy  loam 
suggests  a  possible  influence  of  the  soil  water.  These  results  led  to  a 
further  study  of  the  influence  of  the  soil  moisture  and  of  soil  fertility 
on  the  development  of  the  disease. 


PATHOGENICITY  OF  H.  SATIVUM  31 

INFLUENCE  OF  SOIL  MOISTURE 

Preliminary  series  of  experiments  were  carried  out  in  the  green- 
house in  the  following  manner  in  order  to  determine  the  effect  of  soil 
moisture  on  the  development  of  H .  sativum  on  Lion  barley.  Light  loam 
soil  was  sifted  through  a  5  millimeter  screen,  packed  into  jars,  and 
sterilized.  The  sterilized  soil  was  mixed  with  a  culture  of  H.  sativum 
grown  on  sterilized  oats  seed.  Five  degrees  of  soil  moisture  were  main- 
tained more  or  less  uniformly  by  adding  definite  amounts  of  water  each 
day.  In  the  fifth  series  the  soil  was  kept  saturated  by  standing  the  porous 
pots  in  jars  of  water.  In  the  other  four  series  the  soil  was  in  glazed  jars 
and  the  soil  moisture  was  regulated  by  adding  different  amounts  of 
water.  Each  moisture  series  was  carried  out  in  triplicate,  in  both  inocu- 
lated and  uninoculated  soil.  The  seed  was  sterilized  with  silver  nitrate 
before  planting. 

Comparative  results  on  the  infection  above  ground  at  the  end  of 
three  weeks  and  below  ground  at  the  end  of  four  weeks  are  summarized 
in  Table  X.  In  this  table  the  infection  is  designated  by  fractions;  the 
denominator  represents  the  number  of  plants  in  one  pot,  the  numerator 
the  number  that  were  infected.  On  examining  the  data,  it  is  seen  that, 
as  far  as  the  above  ground  parts  of  the  plants  are  concerned,  the  per- 
centage of  infection,  as  well  as  the  severity,  is  increased  as  the  amount 
of  soil  moisture  is  increased.  Comparatively  few  infections  occurred 
on  the  check  plants. 

The  relation  of  soil  moisture  to  root  infection  is  a  little  more  difficult 
to  see,  as  here  the  development  of  the  roots  in  inoculated  and  uninocu- 
lated soils  with  the  same  moisture  content  must  .be  compared,  and  then 
these  differences  compared  for  the  various  series.  The  roots  were  most 
severely  rotted  in  the  saturated  inoculated  soil,  and  the  difference  in 
the  extent  of  the  root  systems  of  diseased  and  check  plants  was  greatest 
here.  The  next  greatest  difference  was  in  the  first  series,  with  a  soil 
moisture  content  averaging  9  per  cent,  while  the  least  difference  was 
found  in  the  third  and  fourth  series.  In  these  two  series  the  plants 
•grew  best  of  all,  in  both  the  inoculated  and  uninoculated  soils.  Injury 
to  the  roots  is  brought  about  by  rather  limited  local  lesions  which  kill 
the  root  tips  or  cut  off  portions  of  the  roots  when  the  lesions  occur  back 
from  the  tips.  Very  often  the  roots  are  rotted  off  near  the  seed. 

These  results  would  indicate  that  plants  suffer  most  from  root  infec- 
tion by  H.  sativum  in  soils  containing  both  maximum  and  minimum 
extremes  of  moisture.  When  conditions  are  more  nearly  favorable  for 
the  optimum  growth  of  the  plants,  the  effect  of  the  disease  can  be  over- 
come, and  root  systems  are  developed  in  inoculated  soil  almost  equal 
in  extent  to  those  in  clean  soil. 


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36  TECHNICAL  BULLETIN  17 

An  attempt  was  made  to  check  up  the  moisture  relations  of  the 
disease  under  field  conditions.  In  a  series  of  six  square-rod  plots,  three 
were  planted  to  barley  and  three  to  wheat.  The  north  half  of  the  barley 
plots  was  planted  with  Manchuria  and  the  south  half  with  Lion;  the 
north  half  of  the  wheat  plots  was  planted  with  Marquis,  the  south  with 
Bluestem.  A  field  drill  was  used  for  planting.  Several  days  before 
planting,  the  soil  was  inoculated  by  applying  //.  sativum  grown  on 
sterilized  wheat  seed.  One  half  gallon  of  the  culture  was  applied  to 
each  square  rod.  After  the  seed  was  planted,  one  of  the  barley  plots 
and  one  of  the  wheat  plots  was  sprinkled  for  ten  minutes  each  morning 
and  evening,  a  second  of  each  was  sprinkled  for  five  minutes  each  morn- 
ing and  evening,  and  to  the  third  no  water  was  added.  During  the 
first  part  of  the  season,  very  little  infection  appeared  on  any  of  the 
plants  except  Lion  barley.  The  weather  was  very  cold  during  the  first 
two  weeks  after  seeding  and  scarcely  any  infection  occurred.  There 
was  no  seedling  blight  on  the  unwatered  plot,  very  little  on  the  moder- 
ately watered  plot,  and  only  a  moderate  amount  on  the  heavily  watered 
plot.  Infection  occurred  mainly  on  the  above-ground  parts,  resulting 
in  leaf  spots  and  lesions  on  the  sheaths.  Less  than  I  per  cent  of  the 
plants  were  killed.  After  the  first  two  weeks,  the  amount  of  infection 
increased  very  rapidly  on  the  heavily  watered  plot,  so  that  at  the  end 
of  six  weeks  100  per  cent  of  the  plants  were  infected,  and  most  of 
them  were  severely  attacked.  On  the  moderately  watered  plot  about 
80  per  cent  of  the  plants  were  infected,  the  severity  of  the  infection 
ranging  from  light  to  moderate  plus.  On  the  unwatered  plot,  about 
50  per  cent  of  the  plants  were  infected,  the  severity  of  the  infection 
ranging  from  light  to  moderate. 

The  method  of  applying  the  water  tended  to  keep  a  film  of  moisture 
on  the  lower,  shaded  leaves,  forming  almost  a  moist  chamber  near  the 
surface  of  the  soil.  The  sprinkling  also  offered  a  good  opportunity  for 
the  spores  to  be  splashed  from  the  soil  onto  the  leaves.  During  June, 
the  weather  was  very  hot.  As  a  result  of  this  combination  of  circum- 
stances, the  plants  on  the  heavily  watered  plot  were  literally  covered 
with  H.  sativum  lesions.  In  many  cases  the  plants  were  so  badly  in- 
fected at  the  base  that  they  rotted  off.  This  was  not  true  to  such  a 
marked  extent  on  the  moderately  watered  plot  owing,  probably,  to  the 
fact  that  the  surface  of  the  soil  was  not  kept  wet  enough  to  maintain 
a  more  or  less  constant  layer  of  moisture  just  above  the  surface  of  the 
ground.  The  Manchuria  barley  was  moderately  affected,  but  the  wheats 
only  slightly. 


PATHOGENICITY  OF  H.  SATIVUM  37 

Final  data  were  taken  just  before  the  heads  ripened.  An  attempt 
was  made  to  obtain  a  quantitative  estimate  of  the  percentage  of  plants 
infected  for  the  whole  plot  and  an  average  of  the  degree  of  infection 
on  the  roots,  foot,  and  node  from  individual  plants.  For  this  purpose, 
approximately  equally  large  groups  of  plants  were  dug  from  the  center, 
and  also  from  each  corner  of  the  plots,  two  feet  from  the  margins. 
Ten  plants  were  taken  from  each  group  and  for  these  fifty  plants,  the 
following  data  were  recorded  :  the  number  of  culms  which  headed ;  the 
number  of  tillers  which  did  not  head ;  the  degree  of  infection — indicated 
as  heavy,  moderate,  or  light  on  the  roots,  foot,  and  nodes.  Finally,  the 
seed  from  each  plot  was  weighed  after  threshing  and  the  yield  per  acre 
was  calculated  from  this.  These  data  are  summarized  in  Table  XI. 
In  order  to  obtain  a  simple  mathematical  expression  for  making  com- 
parisons, the  percentage  of  heavy  infections  was  multiplied  by  10,  the 
moderate  by  5,  and  the  light  by  i,  and  the  sum  was  taken  as  the  index 
of  the  total  infection.  In  order  more  easily  to  make  comparisons,  these 
sums  were  reduced  to  unity.  Finally,  these  expressions  for  root  and 
foot  infections  were  totaled  to  obtain  a  means  of  comparing  the  com- 
bined foot-  and  root-rot  with  the  relative  amount  of  soil  moisture  and 
with  the  yield.  For  the  sake  of  comparison  these  were  also  reduced  to 
unity.  In  some  cases  the  Helminthosporium  infections  were  so  com- 
plicated by  Fusarinin  infections  that  it  is  quite  impossible  to  say  how 
much  of  the  damage  was  due  to  each  organism.  This  was  especially 
true  on  the  Manchuria  barley.  In  general,  the  amount  of  injury  was 
small.  Altho  the  root-rot  and  the  basal  infection  as  measured  by  the 
degree  of  browning  was  sometimes  heavy  on  a  large  number  of  plants, 
the  plants  were  not  noticeably  stunted  or  immature  as  is  often  the  case 
in  severe  cases  of  foot-rot.  On  the  whole,  there  was  more  foot-  and 
root-rot  on  the  barley  than  on  the  wheat.  The  Lion  barley  alone  shows 
an  increase  in  the  amount  of  foot-rot  as  the  relative  amount  of  soil 
moisture  is  increased.  The  differences  are  so  small,  however,  that  they 
can  not  have  much  significance.  There  were  no  indications  of  a  correla- 
tion in  yield  with  either  the  relative  amount  of  soil  moisture  or  the 
amount  of  foot-  and  root-rot.  For  both  varieties  of  wheat,  foot-  and 
root-rot  was  slightly  worse  on  the  driest  plot.  In  no  case  did  the  rela- 
tive amount  of  soil  moisture  or  infection  influence  the  tillering  of  the 
plants. 


ITIONS 

a 

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PATHOGENICITY  OF  H.  SATIVUM  39 

Many  factors  that  are  difficult  to  control  enter  into  field  experiments 
and  complicate  the  results  in  such  a  way  as  to  make  them  indicative 
rather  than  conclusive.  In  these  experiments  foot-  and  root-rot  de- 
veloped slightly  more  vigorously  on  Lion  barley  in  the  wettest  soil  and 
on  the  two  varieties  of  wheat  in  the  driest  soil.  This  may  be  only  a 
confirmation  of  the  earlier  greenhouse  experience  that  root  infection 
tends  to  be  worse  under  either  extremely  dry  or  extremely  wet  conditions. 

INFLUENCE  OF  SOIL  FERTILITY 

The  effect  of  soil  fertilization  on  the  development  of  foot-  and  root- 
rot  caused  by  H.  sativum  was  studied  in  field  plots  on  Lion  and  Man- 
churia barley,  and  on  Marquis  and  Bluestem  wheat.  Potassium  and 
nitrogen  in  the  form  of  muriate  of  potash  and  nitrate  of  soda  were 
added  to  square-rod  plots  at  the  rate  of  600  and  300  pounds  of  fertilizer 
to  the  acre.  Treble  superphosphate  was  added  at  the  rate  of  200  and 
100  pounds.  These  fertilizers  were  so  applied  that  there  were  plots 
with  a  heavy  and  a  light  application  of  each  alone  and  in  combination 
with  a  heavy  and  light  application  of  each  of  the  others,  except  that 
there  were  no  combinations  of  nitrogen  and  potassium.  In  addition  to 
these,  complete  fertilizer  was  applied  at  the  rate  of  600  and  300  pounds 
and  manure  at  the  rate  of  20  tons  and  10  tons  per  acre.  Unfertilized 
plots  were  left  as  checks.  All  the  plots  were  run  in  duplicate,  one  series 
planted  with  wheat  and  one  with  barley.  The  north  half  of  the  wheat 
plots  was  planted  with  Marquis,  the  south  half  with  Bluestem,  the 
north  half  of  the  barley  plots  with  Lion,  the  south  half  with  Manchuria. 

Several  days  before  planting,  H.  sativum  grown  on  sterilized  wheat 
seed  was  applied  on  the  surface  of  the  soil  at  the  rate  of  one  half  gallon 
of  the  culture  to  the  square  rod.  The  plots  were  seeded  with  a  field 
drill,  wheat  at  the  rate  of  90  pounds  to  the  acre,  and  barley  at  the  rate 
of  86  pounds.  This  is  the  normal  rate  of  seeding  for  this  section  of 
the  country. 

Practically  no  seedling  blight  developed  on  any  of  the  plots.  Leaf 
lesions  and  foot-rot  first  appeared  on  the  barley  during  the  second  and 
third  weeks,  and  soon  after  lesions  developed  also  on  the  wheat. 

There  was  considerable  difference  in  the  vigor  and  height  of  plants 
on  the  different  plots  in  response  to  the  different  fertilizers.  During 
the  latter  part  of  the  season,  there  were  differences  in  the  amount  of 
lodging  on  the  various  plots.  Final  data  on  the  amount  of  foot-  and 
root-rot  were  taken  just  previous  to  the  ripening  of  the  grain.  In  order 
to  obtain  an  approximately  quantitative  expression  for  the  amount  of 
infection  in  each  plot,  50  plants  were  selected  from  each  half  square 
rod,  10  from  eacfi  corner,  two  feet  in  from  the  margins,  and  10  from 
the  center  of  the  plot.  For  each  of  these  plants  the  following  data 


40  TECHNICAL  BULLETIN  17 

were  recorded :  the  number  of  culms  which  developed  heads ;  the  num- 
ber of  tillers  which  did  not  mature;  the  degree  of  infection  (designated 
as  heavy,  moderate,  or  light)  on  the  roots,  the  foot,  and  the  nodes. 
After  harvesting,  the  weight  of  the  straw  and  of  the  threshed  grain 
was  recorded,  and  from  this  the  yield  per  acre  was  calculated.  The 
infection  of.  Lion  barley  was  slightly  more  pronounced  than  on  the  other 
hosts.  These  data  for  Lion  barley  are  summarized  in  Table  XII. 

In  order  to  arrive  at  a  simple  factor  which  would  express  the  total 
infection  for  the  roots,  the  foot,  and  the  nodes  for  a  single  plot  and 
would  also  take  into  account  the  severity  of  infection  as  well  as  the 
percentage  of  plants  infected,  the  number  of  heavy  infections  was 
multiplied  by  10,  the  number  of  moderate  infections  by  5,  and  the  num- 
ber of  light  infections  by  I,  and  the  three  products  were  summed.  This 
was  taken  as  an  arbitrary  index  of  the  infection.  In  order  to  make 
comparison  more  simple,  these  summations  were  reduced  to  unity  by 
dividing  each  by  the  lowest  sum.  This  is  the  factor  designated  as  total 
infection  in  Table  XII.  In  order  to  compare  the  combined  effect  of 
root-  and  foot-rot,  the  summation  for  each  was  added  and  these  sums 
in  turn  were  reduced  to  unity. 

In  order  to  bring  out  the  relation  of  infection  to  fertilizers  and 
yields,  the  arbitrary  indices  of  infection  were  grouped  into  three 
classes  and  the  yields  per  acre  into  three  classes,  and  the  fertilizer  plots 
were  arranged  according  to  their  infection  and  yield  in  the  various 
classes  as  shown  in  Table  XIII.  From  this  summary  table  it  is  quite 
clear  that  the  amount  of  foot-  and  root-rot  is  not  correlated  with  any 
particular  fertilizer. 

The  disease  did  not  appear  in  its  severest  form  on  any  of  the  plots. 
Under  the  conditions  of  this  experiment,  there  was  no  evidence  of 
severe  stunting  of  the  plants  or  of  excessive  tillering. 

COMPARISON  OF  SEVERAL  ROOT-ROT  CAUSING 
ORGANISMS 

In  order  to  obtain  comparative  results  on  the  pathogenic  effect  of 
different  soil  organisms  on  Marquis  wheat  and  Lion  barley,  a  culture 
of  Helminthosporium  isolated  from  the  foot-rot  of  wheat  in  Illinois  by 
F.  L.  Stevens  and  a  culture  of  Fusarmm  culmorum  (W.  Sm.) 
Saccardo  isolated  from  scabby  wheat,  were  compared  with  the  Helmin- 
thosporium sativum  isolated  from  barley  foot-rot  in  Minnesota. 


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TECHNICAL  BULLETIN  17 


These  organisms  were  grown  on  sterilized  wheat  seed  to  obtain  a 
large  amount  of  inoculum.  At  the  end  of  two  months,  the  cultures 
were  practically  masses  of  mycelium  and  spores.  These  masses  were 
passed  through  a  meat  grinder  and  the  pulp  was  thoroly  mixed  with 
sterilized  soil  in  which  the  wheat  and  barley  were  then  planted.  The 
observations  on  seedling  injury  at  the  end  of  twenty  days  are  sum- 
marized in  Table  XIV. 

TABLE   XIII 

SUMMARY  OF  DATA  IN  TABLE  XII 
Infection  classes 


i.o  to  1.3 

1.4  to  1.8                                  1.9  to  2.2 

Yield 

K             N             P 

K             N             P 

K            N             P 

300                        100 

600 

4> 

to   § 

200 

300 

0      ^ 

300                                     200 

No  fertilizer 

06    3 

No  fertilizer 

600                         100 

300          200                             600          200 

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20  tons  manure 
600         600         200 

600 

2  o, 

600          100 

°     3 

CQ 

300         300          100 

300          100 

The  results  on  the  Lion  barley  were  very  sharp.  Unfortunately, 
rats  molested  some  of  the  pots.  In  the  barley  series,  however,  only  the 
check  plants  were  injured.  Three  plants  were  left  in  each  of  the  three 
pots.  The  comparative  size  of  the  plants  grown  in  soil  inoculated  with 
the  various  organisms  is  very  well  shown  by  Plate  IV. 

The  figures  in  Table  XIV  show  that  all  three  of  the  organisms 
caused  a  dwarfing  of  the  barley,  the  two  cultures  of  Helminthosporium 
to  a  much  greater  extent  than  the  Fusarium.  The  Minnesota  strain 
almost  completely  destroyed  the  plants.  The  effect  of  the  three  organ- 
isms on  the  root  systems  is  shown  by  Plate  V.  The  nature  of  the  injury 
caused  by  severe  infections  of  Helminthosporium  is  further  illustrated 
by  Plate  VI,  where  eight  seedlings  showing  various  degrees  of  infection 
are  shown  beside  a  normal  seedling  of  the  same  age  grown  in  sterilized 
soil. 


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44  TECHNICAL  BULLETIN  17 

The  results  obtained  with  Marquis  wheat  are  not  so  clear,  as  all  the 
pots  were  molested  more  or  less.  The  amount  of  injury  caused  by  foot- 
arid  root-rot  was  very  much  less  than  on  the  barley.  The  Illinois  strain 
of  Helminthosporium  seemed  to  cause  slightly  more  injury  than  the 
Minnesota  strain. 

This  series  was  started  in  the  greenhouse  during  warm  weather  early 
in  October  when  the  temperature  in  the  house  was  very  high.  The  sub- 
sequent development  of  the  disease  was  most  interesting.  After  three 
weeks,  the  plants  were  thinned  so  that  only  three  remained  in  each  pot 
except  for  those  inoculated  with  H.  sativum,  culture  82a,  of  which  only 
three  plants  in  each  pot  survived.  These  were  badly  stunted  and  in- 
fected at  the  time.  The  pots  were  kept  next  to  the  outer  west  wall  of 
the  greenhouse,  where  the  temperature  was  always  low  during  winter. 
The  position  of  the  pots  was  changed  periodically  so  that  all  the  plants 
would  have  more  or  less  equal  advantages  as  to  sunlight.  The  plants 
grew  remarkably  well,  and  after  a  few  weeks  scarcely  any  differences 
could  be  detected  between  the  different  series.  The  barley  stooled  ex- 
cessively and  did  not  head  well.  The  wheat  was  very  good.  At  heading 
time,  late  in  April,  there  was  practically  no  difference  between  either 
the  wheat  or  the  barley  plants  grown  in  the  clean  soil  and  in  the  soil 
inoculated  with  the  Minnesota  strain  of  H.  sativum  or  the  Fusarium 
culmorum.  The  wheat  in  the  soil  inoculated  with  the  Illinois  strain  of 
Helminthosporium  was  very  bushy  and  developed  only  one  or  two  heads 
per  pot,  while  the  other  wheats  developed  from  four  to  eight.  The 
Lion  barley  was  also  slightly  poorer  in  the  soil  inoculated  with  the 
Illinois  strain  than  in  the  others.  The  barley  did  not  head  well,  how- 
ever, in  any  case. 

Under  the  conditions  of  this  experiment,  then,  the  Helminthosporium 
caused  more  injury  to  Marquis  wheat  and  Lion  barley  than  the  Fusa- 
rium, both  in  the  seeding  and  the  mature  stages.  While  the  Minnesota 
strain  of  Helminthosporium  caused  decidedly  more  seedling  blight  on 
the  barley,  the  Illinois  strain  caused  slightly  more  stunting  of  the  mature 
plants.  The  Illinois  strain  caused  more  injury  to  the  wheat  at  both 
stages. 

SUMMARY  AND  CONCLUSIONS 

In  recent  years  a  foot-  and  root-rot  of  wheat,  rye,  and  barley  has 
been  serious  in  certain  localities  in  Minnesota.  A  Helminthosporium 
of  the  sativum  type  has  been  constantly  isolated  from  the  diseased 
plants.  In  addition  to  causing  a  foot-  and  root-rot,  the  same  type  of 
organism  attacks  the  leaves  and  stems  and  especially  the  nodes,  glumes, 
and  kernels  of  cereals  and  a  large  number  of  wild  grasses.  A  strain 
of  the  organism  was  isolated  from  a  foot-rot  of  barley.  A  pure  culture 
was  secured  by  isolating  a  single  spore.  The  morphology  of  the  organ- 


PATHOGENICITY  OF  H.  SATIVUM  45 

ism  was  studied  under  various  conditions  with  regard  to  its  specific 
identity.  The  physiology  and  pathogenesis  were  studied  with  special 
reference  to  environmental  conditions  most  favorable  to  the  develop- 
ment of  foot-  and  root-rot. 

The  organism  is  capable  of  causing  disease  symptoms  similar  to 
those  described  by  Pammel,  King  and  Bakke  in  1910.  Discrepancies 
are  found  between  the  spore  measurements  of  this  organism  and  that 
described  by  Pammel,  King  and  Bakke,  but  since  wide  variations  oc- 
curred under  different  conditions  in  a  single-spore  culture  of  the 
organism  studied,  the  similarity  of  disease  symptoms  is  considered 
sufficient  justification  for  considering  the  organism  to  be  Hclminthos- 
porium  satwuni  P.  K.  B. 

Variations  in  the  morphology  of  the  spores  were  found  to  occur 
under  different  conditions  of  growth.  For  spores  as  variable  in  length 
as  those  of  //.  sativum,  it  was  found  necessary  to  measure  500  spores 
in  order  to  obtain  accurate  results. 

On  potato  dextrose  agar,  significant  differences  in  mean  length  of 
the  spores  occur  when  the  organism  is  grown  at  different  temperatures. 
The  shortest  spores  with  a  mean  length  of  55.981^0.35  microns  were 
produced  at  28°  C.  The  longest  spores,  with  a  mean  length  of 
67.32 ±0.55  microns,  were  produced  at  14°  C.  The  difference  between 
the  two  means  is  14  times  the  probable  error  of  the  difference. 

The  greatest  differences  in  length  were  found  between  spores  pro- 
duced on  different  substrata.  At  24°  C.  the  mean  length  of  the  spores 
produced  on  potato  dextrose  was  65.75+0.37  microns,  on  autoclaved 
ripe  barley  heads  67.74=1=0.38  microns,  and  on  green  barley  leaves 
83.14+0.29  microns.  The  difference  between  the  means  of  the  spores 
produced  on  the  agar  and  on  the  leaves  is  37  times  as  great  as  the 
probable  error  of  the  difference. 

The  temperature  relations  of  the  fungus  were  studied  and  it  was 
found  that  the  mycelium  will  grow  at  from  i°  C.  to  37°  C.,  the  optimum 
lying  near  28°.  The  spores  germinated  in  redistilled  water  about  equally 
well  at  temperatures  ranging  from  6°  to  39°,  but  the  length  of  the  germ 
tubes  indicated  that  the  optimum  temperature  is  between  22°  and  32°. 
Germ  tubes  penetrated  the  tissue  of  both  the  coleoptile  and  the  leaf  at 
from  12°  to  34°,  but  severe  infection  occurred  through  a  narrower 
range,  from  22°  to  30°,  the  disease  developing  faster  at  the  higher 
temperatures.  Above  30°,  however,  the  development  of  the  lesions 
seemed  to  be  checked,  altho  they  appeared  very  soon  after  inoculation. 
In  general,  we  may  say  that  rather  high  temperatures  are  most  favor- 
able to  the  growth  of  The  fungus,  to  spore  germination,  to  infection,  and 
to  the  development  of  the  disease. 


46  TECHNICAL  BULLETIN  17 

In  phosphoric  acid-potassium  hydroxide  solutions,  the  spores 
germinated  through  a  wide  range  of  hydrogen-ion  concentrations.  A 
double  optimum  occurred,  both  maxima  falling  on  the  alkaline  side  of 
neutrality  at  pH  8.2  and  pH  9.2.  In  Czapek's  solution  minus  the  sugar, 
the  maximum  germination  occurred  at  pH  6  and  pH  8.  In  general, 
the  spores  germinate  better  in  alkaline  solutions  than  in  acid  solutions. 
The  spores  will  tolerate  high  degrees  of  alkalinity. 

Leaf  infection  increases  directly  with  the  amount  of  moisture 
present.  Greenhouse  experiments  indicate  that  the  effects  of  root  and 
foot  infections  are  more  severe  in  extremely  dry  and  extremely  wet 
soils  than  in  soils  containing  an  optimum  amount  of  moisture  for  the 
growth  of  the  host  plant. 

During  one  year's  field  experimentation,  no  correlation  was  found 
between  the  fertility  of  the  soil  and  the  development  of  foot-  and 
root-rot. 

The  pathogenic  effect  of  H.  sativum  isolated  from  barley  plants  in 
Minnesota  was  compared  with  that  of  a  Helminthosporluni  isolated 
from  stunted  wheat  in  Illinois  and  with  Fusarium  culmorum  isolated 
from  scabby  wheat.  Experiments  were  made  to  determine  the  ability 
of  these  organisms  to  cause  root-  and  foot-rot  of  Marquis  wheat  and 
Lion  barley.  Under  the  conditions  of  the  experiment,  the  Helmin- 
thosporiums  caused  more  injury  than  the  Fusarium.  The  Minnesota 
strain  of  Helminthosporium  caused  the  greater  amount  of  seedling 
injury  on  the  Lion  barley,  while  the  Illinois  strain  caused  the  greater 
dwarfing  of  the  mature  plants  on  both  wheat  and  barley. 

As  a  result  of  these  studies,  the  wide-spread  occurrence  of  H. 
satwum  may  be  explained  by  the  fact  that  the  fungus  responds  sapro- 
phytically  to  such  a  wide  range  of  environmental  conditions.  Neither 
the  effect  of  temperature  nor  acidity  seems  to  be  a  limiting  factor  in  the 
development  of  the  disease  so  far  as  spore  germination  is  concerned. 
As  a  parasite,  the  fungus  causes  rather  limited  local  infections.  The 
amount  of  injury  is  determined  largely  by  the  number  and  size  of  the 
lesions.  A  direct  correlation  exists  between  the  amount  of  moisture 
present  and  the  number  of  lesions.  The  severity  of  the  infection  is 
greater  at  rather  high  temperatures  than  at  low  temperatures.  The 
disease  may  be  expected  to  develop  most  severely,  therefore,  at  high 
temperatures  in  the  presence  of  sufficient  moisture. 

Root  and  foot  infections  are  more  severe  in  certain  soils  than  in 
others.  This  is  probably  largely  due  to  differences  in  soil  moisture 
and  temperature.  In  general,  the  disease  causes  the  greatest  injury 
under  conditions  unfavorable  to  the  growth  of  the  host.  Factors,  such 
as  soil  fertility,  which  might  then  be  expected  to  influence  the  disease, 
apparently  have  little  effect. 


PATHOGENICITY  OP  H.  SATIVUM  47 

LITERATURE  CITED 

1.  Alway,   F.   J.      Agricultural   value   and   reclamation   of    Minnesota   peat   soils. 
Minn.  Agr.  Exp.  Sta.  Bui.   188.     1920.    (Out  of  print.) 

2.  Babcock,    E.    B.    and    Clausen,    R.    E.      Genetics    in    relation    to    agriculture. 
New  York,  1918. 

3.  Christensen,   J.   J.      Studies   on   the   parasitism   of   Hclminthosporium   sativum 
P.K.B.     Master's  Thesis.     Minn.  Agr.  Exp.  Sta.  Tech.  Bui.   11.     1923. 

4.  Clark,  W.  M.     The  determination  of  hydrogen-ions.     Baltimore,  1920. 

5.  Hamblin,  C.  O.     "Foot-rot"  of  wheat  caused  by  the  fungus  Hclminthosporium. 
Agr.  G^z.  N.  S.  W.  33:13-19.     1922. 

6.  Hayes,  H.  K.  and  Stakman,  E.  C.     Resistance  of  barley  to  Hclminthosporium 
sath'um  P.K.B.     Phytopathology  11:405-411.     1921. 

7.  Johnson,  A.  G.    Hclminthosporium  diseases  of  barley  in  Wisconsin.  Phytopath. 

375.     1913- 

8.  McKinney,  H.  H.     The  Hclminthosporium  disease  of  wheat  and  the  influence 
of  soil  temperature  on  seedling  infection.     Phytopath,  12:28.     1922. 

9.  Pammel,  L.  H.,  King,  Charlotte  M.,  and  Bakke,  A.  L.     Two  barley  blights, 
with    comparison   of    species    of    Hclminthosporium   on    cereals.     Iowa    State 
College  of  Agr.  and  Mech.  Arts.     Bui.  116,  June,  1910. 

10.  Schroeder,    H.      Die    Widerstandsfalhigkeit    des    Weizen-und    Gerstenkorns 
gegen  Figte  und  ihre  Becleutung  fuer  die  Sterilisation.     Centralblstt  fiir  Bakt. 
P'arasit.  und  Infekt.  28:492-505.     1910. 

11.  Stakman,  Louise  J.     A  Hclminthosporium  disease  of  wheat  and  rye.     Minne- 
sota Agr.  Exp.  Sta.  Bui.  191.    July,  1920. 

12.  Stevens,  F.  L.      Foot-rot  of  wheat.     Science  N.  S.  51:517-518.     1920. 

13.  —        -  Hclminthosporium  and  wheat   foot-rot.     Phytopathology   n  137.     1921. 

14.  Webb,  Robert  W.     Studies  in  the  physiology  of  the  fungi.  X.    Germination  of 
the  spores  of  certain  fungi  in  relation  to  hydrogen-ion  concentration.     Annals 
Missouri  Botanical  Garden  6:201-222.     1919. 


PLATE  I 

Helminthosporium  sativum  P.  K.  B.  grown  on  potato  dextrose  agar  at 
the  following  temperatures  : 

(i)     o°—   2°C,     (2)     6°  —   8°C,     (3)    13°  —  i5°C, 
(4)    17°  —  22°C,     (5)   2i°  — 24°C.,      (6)   30°  —  32°C., 
(7)   34°-35°C.,     (8)   40°— 42°C. 
Incubated  9  drys. 

PLATE  II 

Helminthospcrium  sativum  P.  K.  B.  grown  on  potato  dextrose  agar 
at  the  following  temperatures: 

(i)     3°-   6°C,     (2)    I2°-I3°C,      (3)    I5°-I8°C, 
(4)   20°  —  23°C.,     (5)   27°—  28°  C,     (6)   31°  —  33°C. 

Incubated  7  days. 

PLATE  III 

Marquis  wheat  showing  the  effect  of  Helminthosporium  root-rot  in 
the  following  types  of  soil : 

A  i.      Heavy  loam,  soil  inoculated  with  H.  sativum  P.K.B. 

A  iC.  Heavy  loam,  uninoculated,  sterilized  soil 

A  2.      Sandy  loam,  soil  inoculated  with  H.  sativum  P.K.B. 

A  2C.  Sandy  loam,  uninoculated,  sterilized  soil 

A  3.      Sand,  soil  inoculated  with  H.  sativum  P.K.B. 

A3C.  Sand,  uninoculated,  sterilized  soil. 

A  4.      Peat,  soil  inoculated  with  H.  sativum  P.K.B. 

A  4C.  Peat,  uninoculated,   sterilized  soil. 

PLATE  IV 

Lion  barley  plants,  3  weeks  old,  growing  in  soil  inoculated  with  the 
following  organisms: 

1.  Helminthosporium  sativum  P.K.B.    Culture  82a,  isolated  from  stunted 

barley  plants  in  Minnesota 

2.  Helminthosporium  isolated  from  stunted  wheat  plants  in  Illinois 

3.  Fusarium  culmorum  (W.  Sm.)   Saccardo 

4.  Uninoculated,  sterilized  soil 

PLATE  V 

Lion  barley  plants,  3  weeks  old,  showing  effect  of  the  following  soil 
organisms  on  development  of  root  systems 

1.  Helminthosporium  sativum  P.K.B.     Culture  82a 

2.  Helminthosporium  from  stunted  wheat  plants  in  Illinois 

3.  Fusarium  culmorum  (W.  Sm.)   Saccardo 

4.  Normal  roots  grown  in  uninoculated,  sterilized  soil 

PLATE  VI 

Lion  barley  plants,  3^2  weeks  old,  showing  effect  of  root  infection  by 
Helminthosporium  sativum  P.K.B.  The  eight  seedlings  on  the  left  were 
grown  in  inoculated  soil,  the  one  on  the  right  in  uninoculated,  sterilized  soil. 


PLATE  I 


PLATE  II 


Louise  Dosdall  was  born  in  Waco,  Texas,  December  n, 
1893.  In  1901  her  parents  moved  to  LeSueur,  Minnesota. 
She  attended  the  public  schools  there,  and  then  finished  the 
grammar  schools  at  St.  Paul,  Minn.  From  1908  to  1912  she 
attended  Humboldt  High  School,  in  St.  Paul. 

In  1912  she  entered  the  College  of  Science,  Literature,  and 
the  Arts  of  the  University  of  Minnesota.  As  an  undergraduate, 
she  majored  in  botany,  specializing  particularly  in  plant  ecology. 
Her  minor  work  was  in  chemistry  and  education.  In  1916  she 
received  the  B.A.  degree.  During  the  following  year  she  acted 
as  teaching  fellow  in  botany  at  Macalester  College,  St.  Paul, 
and  pursued  graduate  work  in  plant  ecology  and  plant  pathology 
at  the  University  of  Minnesota,  receiving  the  M.A.  degree  in 
1917. 

From  June,  1917,  to  July,  1920,  she  was  a  half-time  assistant 
in  plant  pathology  in  the  College  of  Agriculture,  University  of 
Minnesota,  devoting  the  remainder  of  her  time  to  graduate 
work  in  plant  pathology  and  mycology. 

Since  July  1920  she  has  been  an  instructor  in  the  College 
of  Agriculture  and  Mycologist  in  the  Agricultural  Experiment 
Station  of  the  University  of  Minnesota. 


Gaylord  Bros. 

Makers 
Syracuse,  N.  Y. 

PAT.  JAN  21,  1903 


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